Neuropathological Characterization of Dolphin Morbillivirus Infection in Cetaceans Stranded in Italy.

Cetacean morbillivirus (CeMV) is responsible for epidemic and endemic fatalities in free-ranging cetaceans. Neuro-inflammation sustained by CeMV is a leading cause of death in stranded cetaceans. A novel dolphin morbillivirus (DMV) strain of Atlantic origin circulating in Italian waters since early 2016 has caused acute/subacute lesions associated with positive immunolabelling of the virus. To date, myelin damage has not been fully documented and investigated in cetaceans. This study describes neuropathological findings in the brain tissue of 31 cetaceans found stranded along the Italian coastline and positive for DMV infection on molecular testing. Cell changes in the areas of myelinopathy were revealed by double indirect immunofluorescence. The most frequent DMV-associated lesions were astro-microgliosis, neuronal necrosis, spongiosis, malacia, and non-suppurative meningoencephalitis. Myelin reduction and areas of demyelination were revealed by means of a specific myelin biomarker. Morbilliviral antigen immunolabelling was mainly observed in neurons and microglial cells, in association with a marked activation of microglia and astrocytes. These findings extend our knowledge of DMV-associated brain lesions and shed light on their pathogenesis.

Evidence for Unknown Sarcocystis-Like Infection in Stranded Striped Dolphins (Stenella coeruleoalba) from the Ligurian Sea, Italy.

Two striped dolphins (SD1, SD2), stranded along the Ligurian coast of Italy, were diagnosed with a nonsuppurative meningoencephalitis associated with previously undescribed protozoan tissue cysts. As tissue cysts were morphologically different from those of Toxoplasma gondii, additional histopathological, immunohistochemical, ultrastructural, and biomolecular investigations were performed, aiming to fully characterize the organism. Histopathology revealed the presence of large Sarcocystis-like tissue cysts, associated with limited inflammatory lesions in all CNS areas studied. IHC was inconclusive, as positive staining with polyclonal antisera did not preclude cross-reaction with other Sarcocystidae coccidia. Applied to each animal, 11 different PCR protocols precluded a neural infection by Sarcocystis neurona, Sarcocystis falcatula, Hammondia hammondi, and Neospora caninum. T. gondii coinfection was confirmed only in dolphin SD2. Sarcocystis sp. sequences, showing the highest homology to species infecting the Bovidae family, were amplified from SD1 myocardium and SD2 skeletal muscle. The present study represents the first report of Sarcocystis-like tissue cysts in the brain of stranded cetaceans along with the first description of Sarcocystis sp. infection in muscle tissue of dolphins from the Mediterranean basin.

Isolation and molecular characterisation of Halicephalobus gingivalis in the brain of a horse in Piedmont, Italy.

BACKGROUND: A fatal case of meningoencephalitis was reported in a 13-year-old Koninklijk Warmbloed Paard Nederland stallion, suspected of West Nile virus (WNV) infection, in the Piedmont region of Italy. Clinical signs included right head tilt and circling, depression alternated with excitability, fever and lateral strabismus. Combined treatment consisting of dimethylsulfoxide, dexamethasone, sulphonamides and sedative was administered, but because of the poor conditions the horse was euthanatized and submitted for necropsy. RESULTS: At post-mortem examination no skin lesions were observed, all organs appeared normal on gross evaluation and only head and blood samples were further investigated. Neuropathological findings consisted of granulomatous meningoencephalitis and larvae and adult females of Halicephalobus gingivalis were isolated and identified from the digested brain. Frozen brain was submitted to PCR amplification and 220 bp multiple sequence alignment was analysed by Bayesian phylogenetic analysis. CONCLUSIONS: Phylogenetic inference revealed that the isolate belongs to H. gingivalis Lineage 3. WN surveillance can help to deepen our knowledge of horse neurological disorders investigating their causes and incidence. Moreover, it can help to understand the geographic distribution of the H. gingivalis, to unravel epidemiological information, and to estimate risk for humans.

Diets with different lipid contents do not modify the neuronal membrane lipid raft profile in a scrapie murine model.

UNLABELLED: In Transmissible Spongiform Encephalopathies (TSEs), the localization of the prion protein in the neuronal membrane lipid rafts (LR) seems to play a role in sustaining the protein misfolding. Changes in membrane properties, due to altered lipid composition, affect their organization and interaction between lipids and protein therein, and consequently also membrane resident protein functionality; dietary polyunsaturated fatty acids (PUFAs), gangliosides and cholesterol seem to influence these processes. AIMS: In this work, the influence of administration of different feed, able to change the composition of lipid membrane, on the clinical progression of prion disease was studied. MAIN METHODS: The activity of three diets (hyperlipidic with 6% fats; hypolipidic with 0.1% fats; and purified with 4% fats) was tested in CD1 mouse model experimentally infected with RML scrapie strain. Presence and distribution of typical central nervous system (CNS) lesions and deposits of PrP(sc) were evaluated by histopathological analysis and immunohistochemistry. Analysis of lipids was performed in homogenate and insoluble brain fraction of the neuronal membrane rich in LR. KEY FINDINGS: Results show that a diet with a different lipid level has not a significant role in the development of the scrapie disease. All infected mice fed with different diets died in the same time span. Histology, immunohistochemistry, and neuropathological analyses of the infected brains did not show significant differences between animals subjected to different diets. SIGNIFICANCE: Independently of the diet, the infection induced a significant modification of the lipid composition in homogenates, and a less noticeable one in insoluble brain fraction.

Therapeutic effect of CHF5074, a new γ-secretase modulator, in a mouse model of scrapie.

In Transmissible Spongiform Encephalopathies (TSEs) and Alzheimer disease (AD) both misfolding and aggregation of specific proteins represent key features. Recently, it was observed that PrP (c) is a mediator of a synaptic dysfunction induced by Aß oligomers. We tested a novel γ secretase modulator (CHF5074) in a murine model of prion disease. Groups of female mice were intracerebrally or intraperitoneally infected with the mouse-adapted Rocky Mountain Laboratory prions. Two weeks prior infection, the animals were provided with a CHF5074-medicated diet (375 ppm) or a standard diet (vehicle) until they showed neurological signs and eventually died. In intracerebrally infected mice, oral administration of CHF5074 did not prolong survival of the animals. In intraperitoneally-infected mice, CHF5074-treated animals showed a median survival time of 21 days longer than vehicle-treated mice (p < 0.001). In these animals, immunohistochemistry analyses showed that deposition of PrP (Sc) in the cerebellum, hippocampus and parietal cortex in CHF5074-treated mice was significantly lower than in vehicle-treated animals. Immunostaining of glial fibrillary acidic protein (GFAP) in parietal cortex revealed a significantly higher reactive gliosis in CHF5074-treated mice compared to the control group of infected animals. Although the mechanism underlying the beneficial effects of CHF5074 in this murine model of human prion disease is unclear, it could be hypothesized that the drug counteracts PrP (Sc ) toxicity through astrocyte-mediated neuroprotection. CHF5074 shows a pharmacological potential in murine models of both AD and TSEs thus suggesting a link between these degenerative pathologies.

Decrease in pathology and progression of scrapie after immunisation with synthetic prion protein peptides in hamsters.

Effective therapy for prion diseases is currently unavailable. Recently, vaccination was shown to be effective in mouse models of a particular neurodegenerative conditions: Alzheimer's disease (AD). Here, we report that vaccination with synthetic oligopeptides homologous to the hamster (Mesocricetus auratus) prion protein augments survival time in animals infected intraperitoneally with 263K scrapie agent. For each hamster included in the study, prion-specific serum antibodies as well as deposition of pathological prion protein (PrP(res)), glial fibrillary acidic protein (GFAP), and mRNA expression for cytokines (TNF alpha, IL-1beta, IL-10) in brain tissues were evaluated. In immunized animals, increased survival after challenge was associated with a reduction of cerebral lesion, PrP deposition and GFAP expression; in these animals, anti-prion protein peptide antibody levels were increased, and the expression of pro-inflammatory cytokines (TNF alpha and IL-1beta) was reduced. Vaccination could be an effective therapeutic approach to postpone disease onset.