Comparative histopathologic and viral immunohistochemical studies on CeMV infection among Western Mediterranean, Northeast-Central, and Southwestern Atlantic cetaceans

Abstract

Cetacean morbillivirus (CeMV) is a major natural cause of morbidity and mortality in cetaceans worldwide and results in epidemic and endemic fatalities. The pathogenesis of CeMV has not been fully elucidated, and questions remain regarding tissue tropism and the mechanisms of immunosuppression. We compared the histopathologic and viral immunohistochemical features in molecularly confirmed CeMV-infected Guiana dolphins (Sotalia guianensis) from the Southwestern Atlantic (Brazil) and striped dolphins (Stenella coeruleoalba) and bottlenose dolphins (Tursiops truncatus) from the Northeast-Central Atlantic (Canary Islands, Spain) and the Western Mediterranean Sea (Italy). Major emphasis was placed on the central nervous system (CNS), including neuroanatomical distribution of lesions, and the lymphoid system and lung were also examined. Eleven Guiana dolphins, 13 striped dolphins, and 3 bottlenose dolphins were selected by defined criteria. CeMV infections showed a remarkable neurotropism in striped dolphins and bottlenose dolphins, while this was a rare feature in CeMV-infected Guiana dolphins. Neuroanatomical distribution of lesions in dolphins stranded in the Canary Islands revealed a consistent involvement of the cerebrum, thalamus, and cerebellum, followed by caudal brainstem and spinal cord. In most cases, Guiana dolphins had more severe lung lesions. The lymphoid system was involved in all three species, with consistent lymphoid depletion. Multinucleate giant cells/syncytia and characteristic viral inclusion bodies were variably observed in these organs. Overall, there was widespread lymphohistiocytic, epithelial, and neuronal/neuroglial viral antigen immunolabeling with some individual, host species, and CeMV strain differences. Preexisting and opportunistic infections were common, particularly endoparasitism, followed by bacterial, fungal, and viral infections. These results contribute to understanding CeMV infections in susceptible cetacean hosts in relation to factors such as CeMV strains and geographic locations, thereby establishing the basis for future neuro- and immunopathological comparative investigations.

Fig 1. Neuroanatomical distribution and severity of dolphin morbillivirus-associated lesions in striped dolphins (Stenella coeruleoalba) and bottlenose dolphin (Tursiops truncatus) from the Canary Islands (Spain).

The size and density of dots indicates the severity and extent of the lesions. Bar: 1 cm. AcL, anterior cerebellar lobe; H, hypothalamus; Hy, hypophysis; Met, metencephalon; My, myelencephalon; oc, optic chiasm; OccL, occipital lobe; OrL, orbital lobe; PcL, posterior cerebellar lobe T; PL, paralimbic lobe; thalamus; TL, temporal lobe; Ve, vermis. Brain diagram adapted from Oelschlager, H. & Oelschlager, J. (2009).

Fig 2. Cetacean-morbillivirus (CeMV)-associated lesions in central nervous system of striped dolphins (Stenella coeruleoalba), bottlenose dolphins (Tursiops truncatus), and Guiana dolphins (Sotalia guianensis).

(A) Cerebral meningoencephalitis (case 14). The leptomeninges are inflamed and there is increased cellularity in the underlying cortex and subcortical white matter. A large perivascular cuff (arrow) and a few spongy foci (asterisk) are noted. 20x. H&E. Right upper inset: small lymphocytic and plasmacytic perivascular cuff in the cerebral cortex (case 14). 400x. H&E. Right middle inset: thick perivascular cuff at the gray/white matter interface (case 18). 200x. H&E. Right lower inset: degenerating neuron cell body with microglial neuronophagic nodule (case 19). 400x. H&E. (B) Cerebellar meningoencephalitis (case 13). Diffusely, the pia mater, arachnoid, and dura matter are infiltrated by mononuclear inflammatory cells. 40x. H&E. Left inset: mononuclear inflammation at molecular, Purkinje cell, and inner granular cell layer interface with edema, rarefaction, and focal multinucleate giant cell/syncytial cell (MGCS; arrow) (case 19). 200x. H&E. Right inset: Less severely inflamed cerebellar interface area with three degenerating Purkinje cells, one of which displays a viral intranuclear inclusion body (arrow) (case 19). 400x. H&E. (C) Cerebral cortex (case 26). Degenerating swollen neurons and necrotic neurons with intranuclear and intracytoplasmic viral inclusion bodies. 400x. H&E. Right upper inset: Degenerating swollen astrocyte with intracytoplasmic inclusions. 400x. H&E. Right lower inset: Degenerating neuron with intranuclear inclusion body and adjacent necrotic neuron. 400x. H&E. (D) Cerebral cortex (case 26). Marked positive labeling for CeMV-antigen (red) in degenerating neurons and fewer astrocytes (GFAP-brown) (arrow). There is loss of astrocytic processes and cell bodies in this focus. 400x. Double IHC for CeMV and GFAP. (E) Cerebral cortex (case 26). Moderate labeling for CeMV-antigen in neurons and less so in astrocytes (arrow). There is relative preservation of astrocytes in this focus compared to figure (D) but those present have plump nuclei, cytoplasm, and processes. Note astrocytic processes reaching the perivascular space show occasional positivity for CeMV as well. 400x. Double IHC for CeMV and GFAP. (F) Cerebellum (case 1). There is positive labeling for CeMV-antigen in endothelium focally and adjacent neuroglia. 400x. IHC for CeMV. Inset: CeMV positive labeling in neuronal soma and axon hillock of frontal cerebral cortex (case 9). 400x. IHC for CeMV.

Fig 3. Cetacean morbillivirus (CeMV)-associated lesions in lymph nodes and spleen of striped dolphins (Stenella coeruleoalba), bottlenose dolphins (Tursiops truncatus), and Guiana dolphins (Sotalia guianensis).

(A) Pulmonary lymph node (case 27). Severe cortical and paracortical lymphoid depletion. 40x. H&E. Inset: Higher magnification of the squared area in 3A depicting severe cortical lymphoid depletion. 200x. H&E. (B) Mediastinal lymph node (case 13). Paracortical expansion and numerous multinucleate giant cells/syncytia (MGCS). 40x. H&E. (C) Mediastinal lymph node (case 2). There is diffuse morbilliviral antigen in depleted cortex, paracortex, and sinusoidal leukocytes. 20x. IHC for CeMV. Inset: Diffuse morbilliviral labeling, including MGCS, at squared area in 3E. 400x. IHC for CeMV.

Fig 4. Cetacean morbillivirus-associated lesions and comorbidities in lung of striped dolphins (Stenella coeruleoalba), bottlenose dolphins (Tursiops truncatus) and Guiana dolphins (Sotalia guianensis).

(A) Lung (Case 19). Detail of severe inflammation of alveolar spaces and septa with numerous multinucleate giant cells/syncytia (MGCS). Right upper inset: MGCS lining an alveolar septum. 400x. H&E. Right lower inset: pancytokeratin-positive MGCSs lining alveolar septa. 200x. IHC for AE1/AE3. (B) Lung (case 3). Morbilliviral labeling in areas adjacent to occlusive bronchiolar fungal masses (asterisks). 40x. IHC for CeMV. Inset: Epithelial and MGCS labeling in a bronchiole with lamina propria mineralization (asterisks) and luminal adult H. brasiliensis (arrow). 400x. IHC for CeMV.