Cetacean Morbillivirus and Toxoplasma gondii Co-infection in Mediterranean Monk Seal Pup, Italy.

A Mediterranean monk seal (Monachus monachus) pup from the southern Adriatic coast of Italy showed cetacean morbillivirus (CeMV) and disseminated Toxoplasma gondii co-infection, which probably resulted from CeMV-induced immunosuppression. These findings are of concern for the conservation of this critically endangered species.

Disseminated Scedosporium apiospermum infection in a Maremmano-Abruzzese sheepdog.

BACKGROUND: Few cases of scedosporiosis have been reported in animals, but the true prevalence is probably underestimated due to a lack of awareness. Scedosporiosis in dogs has often been associated with localized infection (i.e., nasal infection, eumycetoma, or keratomycosis) or, in rare cases, disseminated infections. CASE PRESENTATION: This case report describes the clinical and pathological features and the diagnostic process of a rare systemic and fatal fungal infection in a dog caused by Scedosporium apiospermum. A 10-month-old female Maremmano-Abruzzese sheepdog showing weakness, lethargy, lateral decubitus, miosis and muscular rigidity was presented. Rodenticide poisoning was clinically suspected for the differential diagnosis. However, postmortem examinations revealed the presence of a swollen and soft subcutaneous nodule located near the right inguinal breast, which was associated with massive enlargement of the inguinal lymph nodes and small disseminated, cream-colored nodules in the kidneys and mesentery. Multiple fungal pyogranulomas were observed upon histological examination. Fungal isolation from the kidneys, breast and inguinal lymph nodes was performed. The internal transcribed spacer (ITS) sequences from the fungal colony DNA were searched in BLAST in the NCBI GenBank for species identification. The sequences of the fungi isolated from the kidney and breast cultures showed 100% sequence identity with sequences from Scedosporium apiospermum. CONCLUSIONS: This report shows that Scedosporium apiospermum may act as a primary pathogen in young and apparently healthy dogs and represents an important pathogen that should be considered during the diagnostic process, particularly when a fungal infection is suspected.

Neurobrucellosis in a common bottlenose dolphin (Tursiops truncatus) stranded in the Canary Islands.

BACKGROUND: Brucella spp. isolation is increasingly reported in cetaceans, although associated pathologies, including lesions of the musculoskeletal and nervous systems, are less frequently described. Concerning the nervous system, Brucella sp. infection causing meningitis, meningoencephalitis or meningoencephalomyelitis have been extensively reported in striped dolphins (Stenella coeruleoalba), and less frequently in other cetacean species. CASE PRESENTATION: A juvenile female common bottlenose dolphin (Tursiops truncatus) was found stranded alive in Lanzarote (Canary Islands, Spain) in 2005, but died shortly after. On physical examination, the dolphin showed a moderate body condition and was classified as code 2 (fresh dead) at the time of necropsy. The main gross findings were severe multiorgan parasitism, thickened and congested leptomeninges, and (sero)fibrino-suppurative and proliferative arthritis of the shoulder joint. Histopathological examination revealed the distinct features of a sub-acute systemic disease associated with Cetacean Morbillivirus (CeMV) infection. However, brain lesions diverged from those reported in systemic CeMV infection. This led to suspect that there was a coinfecting pathogen, based on the characteristics of the inflammatory response and the lesion distribution pattern in the central nervous system. Brucella sp. was detected in the brain tissue by PCR and Brucella antigen was demonstrated by immunohistochemistry in the brain and shoulder joint lesions. CONCLUSIONS: The zoonotic potential of marine mammal strains of Brucella has been demonstrated both in natural and laboratory conditions. In this study, PCR detected Brucella sp. in the brain of a common bottlenose dolphin stranded in the Canary Islands; the dolphin was also co-infected with CeMV. This is the first detection of Brucella sp. infection in a stranded cetacean in this archipelago. Therefore, we stress the importance of taking adequate measures during the handling of these species to prevent the transmissions of the infection to humans.

Host species barriers to Jaagsiekte sheep retrovirus replication and carcinogenesis.

Understanding the factors governing host species barriers to virus transmission has added significantly to our appreciation of virus pathogenesis. Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA), a transmissible lung cancer of sheep that has rarely been found in goats. In this study, in order to further clarify the pathogenesis of OPA, we investigated whether goats are resistant to JSRV replication and carcinogenesis. We found that JSRV induces lung tumors in goats with macroscopic and histopathological features that dramatically differ from those in sheep. However, the origins of the tumor cells in the two species are identical. Interestingly, in experimentally infected lambs and goat kids, we revealed major differences in the number of virus-infected cells at early stages of infection. These differences were not related to the number of available target cells for virus infection and cell transformation or the presence of a host-specific immune response toward JSRV. Indeed, we also found that goats possess transcriptionally active endogenous retroviruses (enJSRVs) that likely influence the host immune response toward the exogenous JSRV. Overall, these results suggest that goat cells, or at least those cells targeted for viral carcinogenesis, are not permissive to virus replication but can be transformed by JSRV.

Pathogen Prevalence in Cetaceans Stranded along the Italian Coastline between 2015 and 2020.

The monitoring of stranded marine mammals represents a strategic method to assess their health, conservation status, and ecological role in the marine ecosystem. Networks worldwide track stranding events for the passive monitoring of mortality patterns, emerging and reemerging pathogens, climate change, and environmental degradation from a One Health perspective. This study summarizes pathogen prevalence data from the Italian Stranding Network (ISN) derived from post-mortem investigations on cetaceans found dead stranded along the Italian coastline between 2015 and 2020. The decomposition of the carcasses and logistics limited the post-mortem examination to 585 individuals, out of 1236 single-stranding reports. The most relevant pathogens identified were Cetacean Morbillivirus, Herpesvirus, Brucella spp., and Toxoplasma gondii, whose roles as environmental stressors are well known, despite their real impact still needing to be investigated in depth. Statistical analysis showed that age and sex seem to be positively related to the presence of pathogens. This study represents the first step in harmonizing post-mortem investigations, which is crucial for evidence-based conservation efforts. Implementing diagnostic and forensic frameworks could offer an indirect insight into the systematic monitoring of diseases to improve the identification of regional and temporal hotspots in which to target specific mitigation, management, and conservation strategies.

Lung adenocarcinoma originates from retrovirus infection of proliferating type 2 pneumocytes during pulmonary post-natal development or tissue repair.

Jaagsiekte sheep retrovirus (JSRV) is a unique oncogenic virus with distinctive biological properties. JSRV is the only virus causing a naturally occurring lung cancer (ovine pulmonary adenocarcinoma, OPA) and possessing a major structural protein that functions as a dominant oncoprotein. Lung cancer is the major cause of death among cancer patients. OPA can be an extremely useful animal model in order to identify the cells originating lung adenocarcinoma and to study the early events of pulmonary carcinogenesis. In this study, we demonstrated that lung adenocarcinoma in sheep originates from infection and transformation of proliferating type 2 pneumocytes (termed here lung alveolar proliferating cells, LAPCs). We excluded that OPA originates from a bronchioalveolar stem cell, or from mature post-mitotic type 2 pneumocytes or from either proliferating or non-proliferating Clara cells. We show that young animals possess abundant LAPCs and are highly susceptible to JSRV infection and transformation. On the contrary, healthy adult sheep, which are normally resistant to experimental OPA induction, exhibit a relatively low number of LAPCs and are resistant to JSRV infection of the respiratory epithelium. Importantly, induction of lung injury increased dramatically the number of LAPCs in adult sheep and rendered these animals fully susceptible to JSRV infection and transformation. Furthermore, we show that JSRV preferentially infects actively dividing cell in vitro. Overall, our study provides unique insights into pulmonary biology and carcinogenesis and suggests that JSRV and its host have reached an evolutionary equilibrium in which productive infection (and transformation) can occur only in cells that are scarce for most of the lifespan of the sheep. Our data also indicate that, at least in this model, inflammation can predispose to retroviral infection and cancer.

Brucella ceti Infection in Striped Dolphins from Italian Seas: Associated Lesions and Epidemiological Data.

Brucella ceti infections have been increasingly reported in cetaceans. In this study, we analyzed all cases of B. ceti infection detected in striped dolphins stranded along the Italian coastline between 2012 and 2021 (N = 24). We focused on the pathogenic role of B. ceti through detailed pathological studies, and ad hoc microbiological, biomolecular, and serological investigations, coupled with a comparative genomic analysis of the strains. Neurobrucellosis was observed in 20 animals. The primary histopathologic features included non-suppurative meningoencephalitis (N = 9), meningitis (N = 6), and meningoencephalomyelitis (N = 5), which was also associated with typical lesions in other tissues (N = 8). Co-infections were detected in more than half of the cases, mostly involving Cetacean Morbillivirus (CeMV). The 24 B. ceti isolates were assigned primarily to sequence type 26 (ST26) (N = 21) and, in a few cases, ST49 (N = 3). The multilocus sequence typing (cgMLST) based on whole genome sequencing (WGS) data showed that strains from Italy clustered into four genetically distinct clades. Plotting these clades onto a geographic map suggests a link between their phylogeny and the topographical distribution. These results support the role of B. ceti as a primary neurotropic pathogen for striped dolphins and highlight the utility of WGS data in understanding the evolution of this emerging pathogen.

Cellular Prion Protein Expression in the Brain Tissue from Brucella ceti-Infected Striped Dolphins (Stenella coeruleoalba).

Brucella ceti, a zoonotic pathogen of major concern to cetacean health and conservation, is responsible for severe meningo-encephalitic/myelitic lesions in striped dolphins (Stenella coeruleoalba), often leading to their stranding and death. This study investigated, for the first time, the cellular prion protein (PrPc) expression in the brain tissue from B. ceti-infected, neurobrucellosis-affected striped dolphins. Seven B. ceti-infected, neurobrucellosis-affected striped dolphins, found stranded along the Italian coastline (6) and in the Canary Islands (1), were investigated, along with five B. ceti-uninfected striped dolphins from the coast of Italy, carrying no brain lesions, which served as negative controls. Western Blot (WB) and immunohistochemistry (IHC) with an anti-PrP murine monoclonal antibody were carried out on the brain parenchyma of these dolphins. While PrPc IHC yielded inconclusive results, a clear-cut PrPc expression of different intensity was found by means of WB analyses in the brain tissue of all the seven herein investigated, B. ceti-infected and neurobrucellosis-affected cetacean specimens, with two dolphins stranded along the Italian coastline and one dolphin beached in Canary Islands also exhibiting a statistically significant increase in cerebral PrPc expression as compared to the five Brucella spp.-negative control specimens. The significantly increased PrPc expression found in three out of seven B. ceti-infected, neurobrucellosis-affected striped dolphins does not allow us to draw any firm conclusion(s) about the putative role of PrPc as a host cell receptor for B. ceti. Should this be the case, an upregulation of PrPc mRNA in the brain tissue of neurobrucellosis-affected striped dolphins could be hypothesized during the different stages of B. ceti infection, as previously shown in murine bone marrow cells challenged with Escherichia coli. Noteworthy, the inflammatory infiltrates seen in the brain and in the cervico-thoracic spinal cord segments from the herein investigated, B. ceti-infected and neurobrucellosis-affected striped dolphins were densely populated by macrophage/histiocyte cells, often harboring Brucella spp. antigen in their cytoplasm, similarly to what was reported in macrophages from mice experimentally challenged with B. abortus. Notwithstanding the above, much more work is needed in order to properly assess the role of PrPc, if any, as a host cell receptor for B. ceti in striped dolphins.

Chlorinated Persistent Organic Pollutants (PCDD/Fs and PCBs) in Loggerhead Sea Turtles Stranded along the Central Adriatic Coast.

Persistent organic pollutants are widespread in the marine environment. They can bioaccumulate and biomagnify in marine organisms through the food web with a potentially toxic effect on living organisms. The sea turtle Caretta caretta is a carnivorous animal with opportunistic feeding behavior. These turtles tend to bioaccumulate pollutants through food, and hence they can be considered an indicator of chemical pollutants in the marine ecosystem. In this study, 44 loggerhead sea turtles were considered, and liver and fat tissue were sampled from each of them to investigate the levels of dioxins (PCDD/Fs) and polychlorinated biphenyls (PCBs) in sea turtles and their potential correlation with sex and size in terms of curved carapace length (CCL). Results suggested that these contaminants were easily bioaccumulated, and PCBs were predominant compared to dioxins in both liver and fat tissue. The congener patterns were similar to those found in sea fish. Moreover, there were no differences in the contamination levels between females and males, nor was there a correlation with the size. There is a need to harmonize the methodological approaches to better evaluate the results and trends over time and to monitor the species and indirectly the health status of the marine environment.

First Report of Septicaemic Listeriosis in a Loggerhead Sea Turtle (Caretta caretta) Stranded along the Adriatic Coast: Strain Detection and Sequencing.

Although there are increasing reports on the prevalence of Listeria monocytogenes in wild species, this is the first case of listeriosis in sea turtle. An adult female Caretta caretta was rescued after being stranded alive along the coast of the Abruzzo region (Italy) in summer 2021. The turtle died in 6 days due to respiratory failure. The necropsy showed widespread organ lesions, such as yellow foci of necrosis in many organs, gastrointestinal erosions, pericarditis, and granulomatous pneumonia. Microbiological and histological analyses were performed on several organs. Listeria monocytogenes was isolated from multiple organs, indicating a case of septicaemic listeriosis, and the genome was sequenced and characterized. All the colonies analysed belonged to the same strain serogroup IVb, ST388, and CC388.

Potential SARS-CoV-2 Susceptibility of Cetaceans Stranded along the Italian Coastline.

Due to marine mammals' demonstrated susceptibility to SARS-CoV-2, based upon the homology level of their angiotensin-converting enzyme 2 (ACE2) viral receptor with the human one, alongside the global SARS-CoV-2 occurrence and fecal contamination of the river and marine ecosystems, SARS-CoV-2 infection may be plausibly expected to occur also in cetaceans, with special emphasis on inshore species like bottlenose dolphins (Tursiops truncatus). Moreover, based on immune and inflammatory responses to SARS-CoV-2 infection in humans, macrophages could also play an important role in antiviral defense mechanisms. In order to provide a more in-depth insight into SARS-CoV-2 susceptibility in marine mammals, we evaluated the presence of SARS-CoV-2 and the expression of ACE2 and the pan-macrophage marker CD68. Aliquots of tissue samples, belonging to cetaceans stranded along the Italian coastline during 2020-2021, were collected for SARS-CoV-2 analysis by real-time PCR (RT-PCRT) (N = 43) and Immunohistochemistry (IHC) (N = 59); thirty-two aliquots of pulmonary tissue sample (N = 17 Tursiops truncatus, N = 15 Stenella coeruleoalba) available at the Mediterranean Marine Mammal Tissue Bank (MMMTB) of the University of Padua (Legnaro, Padua, Italy) were analyzed to investigate ACE2 expression by IHC. In addition, ACE2 and CD68 were also investigated by Double-Labeling Immunofluorescence (IF) Confocal Laser Microscopy. No SARS-CoV-2 positivity was found in samples analyzed for the survey while ACE2 protein was detected in the lower respiratory tract albeit heterogeneously for age, gender/sex, and species, suggesting that ACE2 expression can vary between different lung regions and among individuals. Finally, double IF analysis showed elevated colocalization of ACE2 and CD68 in macrophages only when an evident inflammatory reaction was present, such as in human SARS-CoV-2 infection.

Atypical Toxoplasmosis in a Mediterranean Monk Seal (Monachus monachus) Pup.

The Mediterranean monk seal (Monachus monachus) is the rarest species of pinniped in the world. Necropsy of a Mediterranean monk seal pup that stranded alive on the southern Adriatic Italian coast and died a few hours later revealed co-infection by cetacean morbillivirus (CeMV) and Toxoplasma gondii. Pathological lesions included a multifocal, moderate to severe, necrotizing myocarditis and a diffuse, chronic, moderate interstitial pneumonia with bronchial and bronchiolar epithelial hyperplasia. Lesions of atypical necrotizing arteritis were seen in the aorta and major pulmonary arteries in association with the presence T. gondii organisms. Severe haemorrhagic foci and lesions of non-suppurative meningoencephalitis, together with the presence of protozoal cysts, were seen in the brain. Co-infection of CeMV and T. gondii has not been previously reported in monk seals. The vascular lesions found in this animal can be considered atypical because they have not been reported in other terrestrial or marine mammal species. The disseminated toxoplasmosis associated with the unusual vascular and haemorrhagic brain lesions could be related to the immunosuppressive effects of CeMV infection.

Specific capture and whole-genome phylogeography of Dolphin morbillivirus.

Dolphin morbillivirus (DMV) is considered an emerging threat having caused several epidemics worldwide. Only few DMV genomes are publicly available. Here, we report the use of target enrichment directly from cetacean tissues to obtain novel DMV genome sequences, with sequence comparison and phylodynamic analysis. RNA from 15 tissue samples of cetaceans stranded along the Italian and French coasts (2008-2017) was purified and processed using custom probes (by bait hybridization) for target enrichment and sequenced on Illumina MiSeq. Data were mapped against the reference genome, and the novel sequences were aligned to the available genome sequences. The alignment was then used for phylogenetic and phylogeographic analysis using MrBayes and BEAST. We herein report that target enrichment by specific capture may be a successful strategy for whole-genome sequencing of DMV directly from field samples. By this strategy, 14 complete and one partially complete genomes were obtained, with reads mapping to the virus up to 98% and coverage up to 7800X. The phylogenetic tree well discriminated the Mediterranean and the NE-Atlantic strains, circulating in the Mediterranean Sea and causing two different epidemics (2008-2015 and 2014-2017, respectively), with a limited time overlap of the two strains, sharing a common ancestor approximately in 1998.

Occurrence of Brucella ceti in striped dolphins from Italian Seas.

Brucella ceti infections have been increasingly reported in cetaceans, although a very limited characterization of Mediterranean Brucella spp. isolates has been previously reported and relatively few data exist about brucellosis among cetaceans in Italy. To address this gap, we studied 8 cases of B. ceti infection in striped dolphins (Stenella coeruleoalba) stranded along the Italian coastline from 2012 to 2018, investigated thanks to the Italian surveillance activity on stranded cetaceans. We focused on cases of stranding in eastern and western Italian seas, occurred along the Apulia (N = 6), Liguria (N = 1) and Calabria (N = 1) coastlines, through the analysis of gross and microscopic findings, the results of microbiological, biomolecular and serological investigations, as well as the detection of other relevant pathogens. The comparative genomic analysis used whole genome sequences of B. ceti from Italy paired with the publicly available complete genomes. Pathological changes consistent with B. ceti infection were detected in the central nervous system of 7 animals, showing non-suppurative meningoencephalitis. In 4 cases severe coinfections were detected, mostly involving Dolphin Morbillivirus (DMV). The severity of B. ceti-associated lesions supports the role of this microbial agent as a primary neurotropic pathogen for striped dolphins. We classified the 8 isolates into the common sequence type 26 (ST-26). Whole genome SNP analysis showed that the strains from Italy clustered into two genetically distinct clades. The first clade comprised exclusively the isolates from Ionian and Adriatic Seas, while the second one included the strain from the Ligurian Sea and those from the Catalonian coast. Plotting these clades onto the geographic map suggests a link between their phylogeny and topographical distribution. These results represent the first extensive characterization of B. ceti isolated from Italian waters reported to date and show the usefulness of WGS for understanding of the evolution of this emerging pathogen.

Transplacental transmission of the Italian Bluetongue virus serotype 2 in sheep

In order to study the capability of a Bluetongue virus serotype 2 (BTV­2) field isolate to cross the placental barrier, 2 groups of 5 pregnant ewes were infected with a field BTV­2 Italian strain (Group A) or with the same strain passaged once in Culicoides cells (Kc) (Group B). Following infection, EDTA­blood and serum samples were collected weekly and tested for the presence of BTV RNA/infectious virus and anti­BTV­2 antibodies, respectively. At lambing, precolostral EDTA­blood and serum samples were collected from lambs and tested as before. The lambs were then sampled as scheduled for the dams. All sheep seroconverted on day 12 post­infection (pi) and remained seropositive throughout the sampling period (day 68 pi). BTV was isolated from day 7 pi to day 14 pi in animals of Group A and from day 5 pi to day 12 pi in animals of Group B. None of the 14 lambs born had pre­colostral antibodies. Three lambs born from two ewes of Group B were viraemic at birth and in one lamb infectious virus was isolated from blood up to 11 days of age. This study proved for the first time that a single passage of BTV­2 field strain in Kc cells is able to give to BTV the ability to cross the placenta barrier and infect foetal tissues.

Spartium junceum L. poisoning in small ruminants.

An outbreak of neurological disorders in a flock of 20 sheep coming from a rural farm in Civitella Roveto, Italy, occurred in winter 2015. All the animals showed tonic-clonic convulsions followed by muscle paralysis associated with dilated pupils, tremor, tachycardia, tachypnea and diarrhea. The presence of bundles of dry broom of Spartium junceum L. in the feed, eaten by the animals supported the hypothesis of plant intoxication. Two animals died after worsening of clinical signs. The anatomopathological findings and the laboratory results ruled out viral or bacterial infections or accidental exposure to other toxics. Phytochemical study showed the presence of large amount of cytisine, a nicotinic acetylcholine receptor agonist, in all parts of the plant eaten by the animals. Clinical and pathological findings, the complete remission of clinical signs after the exclusion of dry broom from the diet, together with the results of phytochemical analyses results corroborated the hypothesis of S. junceum L. intoxication.

Immunohistochemical investigations on Brucella ceti-infected, neurobrucellosis-affected striped dolphins (Stenella coeruleoalba).

Bacteria of the genus Brucella cause brucellosis, an infectious disease common to humans as well as to terrestrial and aquatic mammals. Since 1994 several cases of Brucella spp. infection have been reported in marine mammals worldwide. While sero-epidemiological data suggest that Brucella spp. infection is widespread globally, detecting Brucella spp.-associated antigens by immunohistochemistry (IHC) in tissues from infected animals is often troublesome. The present study was aimed at investigating, by means of IHC based upon the utilization of an anti-Brucella LPS monoclonal antibody (MAb), the central nervous system (CNS) immunoreactivity shown by B. ceti-infected, neurobrucellosis-affected striped dolphins. The aforementioned MAb, previously characterized by means of ELISA and Western Blotting techniques, was able to immunohistochemically detect smooth brucellae both within the CNS from B. ceti-infected striped dolphins and within a range of tissues from Brucella spp.-infected domestic ruminants. In conclusion, the results of the present study are of relevance both from the B. ceti infection's diagnostic and pathogenetic standpoints.

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

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.

Comparative Immunopathology of Cetacean morbillivirus Infection in Free-Ranging Dolphins From Western Mediterranean, Northeast-Central, and Southwestern Atlantic.

Cetacean morbillivirus (CeMV; Paramyxoviridae) causes epizootic and interepizootic fatalities in odontocetes and mysticetes worldwide. Studies suggest there is different species-specific susceptibility to CeMV infection, with striped dolphins (Stenella coeruleoalba), bottlenose dolphins (Tursiops truncatus), and Guiana dolphins (Sotalia guianensis) ranking among the most susceptible cetacean hosts. The pathogenesis of CeMV infection is not fully resolved. Since no previous studies have evaluated the organ-specific immunopathogenetic features of CeMV infection in tissues from infected dolphins, this study was aimed at characterizing and comparing immunophenotypic profiles of local immune responses in lymphoid organs (lymph nodes, spleen), lung and CNS in CeMV-molecularly (RT-PCR)-positive cetaceans from Western Mediterranean, Northeast-Central, and Southwestern Atlantic. Immunohistochemical (IHC) analyses targeted molecules of immunologic interest: caspase 3, CD3, CD20, CD57, CD68, FoxP3, MHCII, Iba1, IFNγ, IgG, IL4, IL10, lysozyme, TGFß, and PAX5. We detected consistent CeMV-associated inflammatory response patterns. Within CNS, inflammation was dominated by CD3+ (T cells), and CD20+ and PAX5+ (B cells) lymphocytes, accompanied by fewer Iba1+, CD68+, and lysozyme+ histiocytes, mainly in striped dolphins and bottlenose dolphins. Multicentric lymphoid depletion was characterized by reduced numbers of T cells and B cells, more pronounced in Guiana dolphins. Striped dolphins and bottlenose dolphins often had hyperplastic (regenerative) phenomena involving the aforementioned cell populations, particularly chronically infected animals. In the lung, there was mild to moderate increase in T cells, B cells, and histiocytes. Additionally, there was a generalized increased expression of caspase 3 in lymphoid, lung, and CNS tissues. Apoptosis, therefore, is believed to play a major role in generalized lymphoid depletion and likely overt immunosuppression during CeMV infection. No differences were detected regarding cytokine immunoreactivity in lymph nodes, spleen, and lung from infected and non-infected dolphins by semiquantitative analysis; however, there was striking immunoreactivity for IFNγ in the CNS of infected dolphins. These novel results set the basis for tissue-specific immunophenotypic responses during CeMV infection in three highly susceptible delphinid species. They also suggest a complex interplay between viral and host's immune factors, thereby contributing to gain valuable insights into similarities, and differences of CeMV infection's immunopathogenesis in relation to body tissues, CeMV strains, and cetacean hosts.

A real-time RT-PCR assay for molecular identification and quantitation of feline morbillivirus RNA from biological specimens.

The aim of this study was to develop a real-time RT-PCR to detect and quantitate feline morbillivirus (FeMV) RNA in biological samples. Primers and probe were targeted on a conserved region of FeMV P/V/C gene. To validate the assay with field samples, a total number of specimens of cats have been recruited including 264 urine and blood samples and compared with a generic RT-PCR targeting the L protein encoding gene of morbilliviruses. In addition, 385 tissue samples from 35 carcasses of cats have been also employed. RNA titres were low in all tested samples. Results also indicated the absence of cross-reaction with related morbilliviruses and existing pathogens of cats. In tissues with low levels of FeMV RNA, the presence of viral antigen was also evidenced by immunohistochemistry targeting the N viral protein. This newly described assay allows for a rapid, accurate and reliable quantitative detection of FeMV RNA that can be applied for diagnostics and research studies.