Introduction

The impact of Morbillivirus infections (MI) on the health status of sea mammals is well established. Indeed, since 1987, at least ten different MI epidemics have caused mass mortality within several free-living pinnipeds and cetaceans populations around the world.¹ Among the still debated, or even controversial issues regarding MI in sea mammals, the one related to the origin of their causative agents is of particular concern, while another intriguing question refers to the synergistic effects, if any, associated with chronic exposure to a number of environmental pollutants, such as organochlorines and heavy metals.² In fact, it is also unknown whether and how these chemicals contribute towards modulating the pathogenic and pathogenetic activity primarily displayed by sea mammal morbilliviruses. Among others, heavy metals have proved to act as immunomodulators in many mammalian species, mainly acting as immunosuppressors.³

Materials and Methods

In present study we report about heavy metals (Cd, Pb and Hg) concentrations in liver and muscle tissue from nine striped dolphins (Stenella coeruleoalba) which were found stranded lifeless, from August to November 2007, on the Ligurian Sea coast of Italy. Sampled cohort included one calf of no more than two weeks of age, two sub-adults and six adult animals.

A detailed post-mortem examination was carried out on all nine dolphins under study, the preservation degree of which allowed us to perform both histopathological and microbiological investigations from each animal's major organs. Furthermore, detailed serological, immunohistochemical (IHC) indirect immunofluorescence (IIF), and biomolecular (reverse transcriptase-polymerase chain reaction, RT-PCR) investigations were carried out on a number of tissues from eight dolphins, in order to assess whether a Morbillivirus infection might have been responsible for their death and subsequent stranding.

More in detail, a serum neutralization test was performed with the "Onderstepoort" Canine Distemper Virus (CDV) strain as antigen, while for IHC and IIF investigations a monoclonal antibody (MoAb) against CDV nucleoprotein (NP) antigen was used, and different sets of primers specifically designed for known genomic regions of Morbillivirus and Dolphin Morbillivirus (DMV) being utilized in RT-PCR studies. Any possible correlation with immunosuppressive effects of heavy metals also was investigated, even if the low number of animals did not allow any statistical analysis.

Results and Discussion

Mean metals concentrations measured in the nine dolphins under study are reported in Table 1. The lowest levels of all metals were found in the calf, but it should be also noted that 10 out of 18 samples were below the limit of detection (LOD) for lead.

As for anatomo-histopathological findings, a subacute to chronic broncho-pneumonia and/or broncho-interstitial pneumonia was observed in six animals, which also showed (similarly to the remaining three dolphins) simultaneous evidence of different parasite organisms variously distributed throughout their body, at both pulmonary and extra-pulmonary locations. Interestingly, a subacute to chronic, multifocal, non-purulent meningo-encephalitis (pan-encephalitis) occurred in four adult animals, with lesions being characterized, among others, by a more or less prominent perivascular edema, peri/paravascular macrophages accumulations with a more or less developed astrogliosis/astrocytosis and mild choroid plexus infiltration of lymphocytes and plasma cells.

Table 1. Mean metals concentrations ± e.s., minimum and maximum, in liver and muscle tissue of the 9 stranded dolphins under study.

More or less prominent haemorrhagic foci could be also observed within both the sub-arachnoidal space and the brain parenchyma, with scattered microglial cells nodules often accompanied by haemosiderin deposits being detected in the grey matter, especially in proximity to blood vessel, which frequently appeared to be newly formed. Mild to moderate neuronal degeneration (peripheral chromatolysis) could be also noted, with no evidence of viral inclusions being found in any brain cell (nor in any other body cell type).

IHC, IIF and RT-PCR investigations for Morbillivirus, which were carried out on the brain, lung and spleen tissue from eight out of the nine dolphins investigated herein, yielded negative results, despite the presence of anti-CDV neutralizing antibodies in the blood serum of four animals, which showed Morbillivirus-positive titres of 1:10 (calf), 1:20 (one sub-adult and one adult female) and 1:40 (one adult female), respectively.

On the basis of the above results, it seems more than realistic to believe that exposure to a morbilliviral agent took place in at least four of the nine dolphins under study, which were found stranded along a short coastal trait (Imperia Province, Ligurian Sea/Northern Tyrrhenian Sea) and across a limited time frame (August-November 2007). Indeed, the Morbillivirus epidemic currently affecting Mediterranean Sea striped dolphins, whose "index cases" might have been the DMV-infected pilot-whales (Globicephala melas) which were reported around Gibraltar between the end of 2006 and the early months of 2007⁴, is a matter of special concern.⁵ This is particularly true at the light of the dramatic morbilliviral epidemic occurred between 1990 and 1992 in the same area, which caused the death of hundreds, if not thousands, of Mediterranean striped dolphins.^1,2,6

Nevertheless, despite the seropositivity clearly documented in four animals investigated herein, that could supposedly argue in favour of their recent exposure to the morbilliviral strain(s) currently circulating in the Mediterranean basin (considering also that such dolphins were of different ages, including the newborn animal whose 1:10 seropositive titre may have likely resulted from passive transfer maternal immunity to the virus), both IHC, IIF and RT-PCR investigations against Morbillivirus yielded negative results in all eight cetaceans on which they were carried out, including the four adult dolphins affected with inflammatory brain tissue changes compatible--at least in part--with a morbilliviral encephalitis.^1,2,6

As far as metal levels are concerned, they were always below the immunotoxic thresholds for cadmium and lead, while constantly being well over the threshold for mercury. Therefore, a certain degree of immunosuppression, including an increased rate of immune cells apoptosis, along with a reduction in lymphocyte proliferation and phagocytic activity, could be considered for all animals.³

Interestingly, when metal data were evaluated on the basis of the results of serological investigations, higher levels of metals were found in seronegative animals, as clearly documented for hepatic lead and mercury (Figure 1a-b).

Click on the image to see a larger view.

Figure 1. Mean levels of lead (Pb; Panel a) and mercury (Hg; Panel b) in the liver tissue of the nine stranded dolphins in comparison with the results of serological investigations (serum neutralization test) against Morbillivirus ("Onderstepoort" CDV strain).

In conclusion, the data obtained in the present study suggest that, albeit (present or past) exposure to a morbilliviral agent was clearly documented in four dolphins, one or more factors other than Morbillivirus could have been involved in the development of lesions and the animal's subsequent death. This may be also the case of subacute to chronic meningo-encephalitis, showing similarities with those reported in the course of Brucella spp. infection in striped dolphins⁷. In this respect, the immunotoxic effect displayed by mercury and presumably other environmental contaminants in cetaceans included in this study may have exacerbated the pathogenic activity of one or more biologic disease agents.

Further and more-in-depth investigations are needed to better define the adverse roles of pollutants for sea mammals' health and conservation and their role within the context of the past (1990-1992) and the present morbilliviral epidemics in Mediterranean striped dolphins.

Acknowledgements

We are very grateful to Dr. Seamus Kennedy for providing us with the positive control tissue samples utilized in IHC and IIF investigations. This work was partially carried out with a research grant from Ministero dell'Ambiente e della Tutela del Territorio e del Mare (Progetto di Ricerca "Cause di Mortalità e Studi Patogenetici in Cetacei Spiaggiati sulle Coste Italiane").

References

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2.  Di Guardo G, Marruchella G, Agrimi U, Kennedy S. 2005. Morbillivirus infections in aquatic mammals: A brief review. J. Vet. Med. A 52:88-93.

3.  Camara-Pellisso S, Munoz MJ, Carballo M, Sanchez-Vizcaino JM. 2008. Determination of the immunotoxic potential of heavy metals on the functional activity of bottlenose dolphin leukocytes in vitro. Vet. Immunol. Immunopathol. 121:189-198.

4.  Fernández A, Esperon F, Herraez P, Espinoza de Los Monteros A, Clavel C., Bernabè A, Sanchez-Vizcaino JM, Verborgh P, DeStephanis R, Toledano F, Bayon A. 2008. Pilot whales (G. melas) mortality due to Morbillivirus in Mediterranean Sea. Emerg. Infect. Dis. (in press).

5.  Raga J-A, Banyard A, Domingo M, Corteyn M, Van Bressem M-F, Fernández M, Aznar F-J, Barrett T. 2008. Dolphin Morbillivirus epizootic resurgence, Mediterranean Sea. Emerg. Infect. Dis. 14:471-473.

6.  Domingo M, Visa J, Fumarola M, Marco AJ, Ferrer L, Rabanal R, Kennedy S. 1992. Pathologic and immunocytochemical studies of Morbillivirus infection in striped dolphins (Stenella coeruleoalba).Vet Pathol. 29:1-10.

7.  González L, Patterson IA, Reid RJ, Foster G, Barberan M, Blasco JM, Kennedy S, Howie FE, Godfroid J, MacMillan AP, Schock A, Buxton D. 2002. Chronic meningoencephalitis associated with Brucella sp. infection in live-stranded striped dolphins (Stenella coeruleoalba). J. Comp. Pathol. 126:147-152.