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Herpesvirus (HV) is widely distributed among cetacean populations, with the highest prevalence reported in the Mediterranean Sea. In this study, a comprehensive analysis was conducted, including epidemiological, phylogenetic, and pathological aspects, with particular emphasis on neuropathology, to better understand the impact of HV in these animals. Our results show a higher presence of HV in males compared to females, with males exhibiting a greater number of positive tissues. Additionally, adults were more frequently affected by HV infection than juveniles, with no infections detected in calves or neonates. The affected species were striped (Stenella coeruleoalba) and bottlenose dolphins (Tursiops truncatus). The highest positivity rates were observed in the genital system, cerebrum, and skin tissues. Phylogenetic analysis indicated a higher occurrence of Gammaherpesvirus (GHV) sequences but increased genetic diversity within Alphaherpesvirus (AHV). Key neuropathological features included astro-microgliosis (n = 4) and meningitis with minimal to mild perivascular cuffing (n = 2). The presence of concurrent infections with other pathogens, particularly cetacean morbillivirus (CeMV), underscores the complex nature of infectious diseases in cetaceans. However, the presence of lesions at the Central Nervous System (CNS) with molecular positivity for GHV, excluding the involvement of other potential neurotropic agents, would confirm the potential of this HV subfamily to induce neurological damage. Pathological examination identified lesions in other organs that could potentially be associated with HV, characterized by lymphoid depletion and tissue inflammation. These findings enhance our understanding of HV in odontocetes and highlight the need for ongoing research into the factors driving these infections and their broader implications., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Vargas-Castro et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

We characterized novel coronaviruses detected in US bottlenose dolphins (BdCoVs) with diarrhea. These viruses are closely related to the other 2 known cetacean coronaviruses, Hong Kong BdCoV and beluga whale CoV. A deletion in the spike gene and insertions in the membrane gene and untranslated regions were found in US BdCoVs (unrelated to severe acute respiratory syndrome coronavirus 2).

Cetaceanpox viruses (CePVs) are associated with a cutaneous disease in cetaceans often referred to as "tattoo" lesions. To date, only partial genomic data are available for CePVs, and thus, they remain unclassified members of the subfamily Chordopoxvirinae within the family Poxviridae. Herein, we describe the first complete CePV genome sequenced from the tattoo lesion of a managed Indo-Pacific bottlenose dolphin (Tursiops aduncus), using next-generation sequencing. The T. aduncus CePV genome (CePV-TA) was determined to encode 120 proteins, including eight genes unique to the CePV-TA and five genes predicted to function as immune-evasion genes. The results of CePV-TA genetic analyses supported the creation of a new chordopoxvirus genus for CePVs. The complete sequencing of a CePV represents an important first step in unraveling the evolutionary relationship and taxonomy of CePVs, and significantly increases our understanding of the genomic characteristics of these chordopoxviruses., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Free-ranging Atlantic bottlenose dolphins ( n = 360) from two southeastern U.S. estuarine sites were given comprehensive health examinations between 2003 and 2015 as part of a multi-disciplinary research project focused on individual and population health. The study sites (and sample sizes) included the Indian River Lagoon (IRL), Florida, USA ( n = 246) and Charleston harbor and associated rivers (CHS), South Carolina, USA ( n = 114). Results of a suite of clinicoimmunopathologic tests revealed that both populations have a high prevalence of infectious and neoplastic disease and a variety of abnormalities of their innate and adaptive immune systems. Subclinical infections with cetacean morbillivirus and Chlamydiaceae were detected serologically. Clinical evidence of orogenital papillomatosis was supported by the detection of a new strain of dolphin papillomavirus and herpesvirus by molecular pathology. Dolphins with cutaneous lobomycosis/lacaziasis were subsequently shown to be infected with a novel, uncultivated strain of Paracoccidioides brasiliensis , now established as the etiologic agent of this enigmatic disease in dolphins. In this review, innate and adaptive immunologic responses are compared between healthy dolphins and those with clinical and/or immunopathologic evidence of infection with these specific viral, bacterial, and fungal pathogens. A wide range of immunologic host responses was associated with each pathogen, reflecting the dynamic and complex interplay between the innate, humoral, and cell-mediated immune systems in the dolphin. Collectively, these studies document the comparative innate and adaptive immune responses to various types of infectious diseases in free-ranging Atlantic bottlenose dolphins. Evaluation of the type, pattern, and degree of immunologic response to these pathogens provides novel insight on disease immunopathogenesis in this species and as a comparative model. Importantly, the data suggest that in some cases infection may be associated with subclinical immunopathologic perturbations that could impact overall individual and population health.

We report the discovery of the first cetacean pegivirus (family Flaviviridae) using a next-generation sequencing approach. One of two infected bottlenose dolphins had elevated activities of liver enzymes, which may suggest hepatocellular injury. Further research is needed to determine the epidemiology and pathogenicity of dolphin pegivirus., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist.

Fecal samples collected from free-ranging Atlantic bottlenose dolphins (BDs) in the Indian River Lagoon of Florida were processed for viral discovery using a next-generation sequencing (NGS) approach. A 693-bp contig identified in the NGS data was nearly identical to the partial L1 gene sequence of a papillomavirus (PV) previously found in a penile papilloma in a killer whale (Orcinus orca). Based on this partial bottlenose dolphin papillomavirus (BDPV) sequence, a nested inverse PCR and primer-walking strategy was employed to generate the complete genome sequence. The full BDPV genome consisted of 7299 bp and displayed a typical PV genome organization. The BDPV E6 protein contained a PDZ-binding motif, which has been shown to be involved in carcinogenic transformation involving high-risk genital human PVs. Screening of 12 individual fecal samples using a specific endpoint PCR assay revealed that the feces from a single female BD displaying a genital papilloma was positive for the BDPV. Genetic analysis indicated that this BDPV (Tursiops truncatus papillomavirus 8; TtPV8) is a new type of Dyopipapillomavirus 1, previously sequenced from an isolate obtained from a penile papilloma in a harbor porpoise (Phocoena phocoena). Although only a partial L1 sequence has been determined for a PV detected in a killer whale genital papilloma, our finding of a nearly identical sequence in an Atlantic BD may indicate that members of this viral species are capable of host jumping. Future work is needed to determine if this virus is a high-risk PV that is capable of inducing carcinogenic transformation and whether it poses a significant health risk to wild delphinid populations.

Clinical and epidemiological features of tattoo skin disease (TSD) are reported for 257 common bottlenose dolphins held in 31 facilities in the Northern Hemisphere. Photographs and biological data of 146 females and 111 males were analyzed. Dolphins were classified into three age classes: 0-3 years, 4-8 years, and older than 9 years. From 2012 to 2014, 20.6% of the 257 dolphins showed clinical TSD. The youngest dolphins with tattoo lesions were 14 and 15 months old. TSD persisted from 4 to 65 months in 30 dolphins. Prevalence varied between facilities from 5.6% to 60%, possibly reflecting variation in environmental factors. Unlike in free-ranging Delphinidae, TSD prevalence was significantly higher in males (31.5%) than in females (12.3%). Infection was age-dependent only in females. Prevalence of very large tattoos was also higher in males (28.6%) than in females (11.1%). These data suggest that male T. truncatus are more vulnerable to TSD than females, possibly because of differences in immune response and susceptibility to captivity-related stress.

The poxviruses identified in cetaceans are associated with characteristic tattoo or ring skin lesions. However, little is known regarding the prevalence and progression of these lesions and the molecular characterization of cetacean poxviruses in the Southern Hemisphere. This manuscript describes the progression of poxvirus-like skin lesions in 5 free-ranging Guiana dolphins Sotalia guianensis. Additionally, 151 skin samples from 113 free-ranging cetaceans from Brazil, including 4 animals with tattoo skin lesions, were selected for poxvirus testing. Poxviral DNA polymerase gene PCR amplification was used to detect the virus in β-actin-positive samples (145/151). DNA topoisomerase I gene PCR was then used in Cetaceanpoxvirus (CePV)-positive cases (n = 2), which were further evaluated by histopathology and electron microscopy. Based on photo-identification, adult Guiana dolphins presented regressing or healed poxvirus-like lesions (2/2), while juveniles presented persistent (2/3) or healed and progressive lesions (1/3). CePV DNA was amplified in a common bottlenose dolphin Tursiops truncatus and in a Guiana dolphin. Intracytoplasmic inclusion bodies and viral particles consistent with poxvirus were identified by histology and electron microscopy, respectively. CePV-specific amino acid motifs were identified through phylogenetic analysis. Our findings corroborate previous studies that suggest the placement of poxviruses from cetaceans within the novel CePV genus. This is the first molecular identification of poxvirus in South American odontocetes.

:  Adenoviruses are nonenveloped, double-stranded DNA viruses, known to infect members of all tetrapod classes, with a similarity between phylogenies of hosts and viruses observed. We characterized bottlenose dolphin adenovirus 2 (BdAdV-2) found in a bottlenose dolphin ( Tursiops truncatus) with enteritis. Virions were seen by negative staining electron microscopy of feces. Initial sequences obtained using conserved PCR primers were expanded using primer walking techniques, and the complete coding sequence was obtained. Phylogenetic analyses were consistent with coevolution of this virus and its bottlenose dolphin host, placing BdAdV-2 into a monophyletic group with other mastadenoviruses of Cetartiodactyla. When considering the low guanine/cytosine (G/C) content of BdAdV-2 with the phylogenetic data, this virus may represent a host-jumping event from another member of Cetartiodactyla. Analysis of partial polymerase indicated that bottlenose dolphin adenovirus 1, previously identified in Spain, and BdAdV-2 are sister taxa with harbor porpoise adenovirus 1, forming a cetacean clade. Bottlenose dolphin adenovirus 2 includes a highly divergent fiber gene. Two genes homologous to the dUTPase superfamily are also present which could play a role in enabling viral replication in nondividing cells. We used sequence data to develop a probe hybridization quantitative PCR assay specific to BdAdV-2 with a limit of detection of 10 copies.

Adenoviruses are common pathogens in vertebrates, infecting a wide range of hosts, but only having rarely been detected and correlated with disease in cetaceans. This article describes the first complete genomic sequence of a cetacean adenovirus, bottlenose dolphin adenovirus 1 (BdAdV-1), detected in captive bottlenose dolphin population (Tursiops truncatus) suffering from self-limiting gastroenteritis. The complete genome sequence of BdAdV-1 was recovered from data generated by high-throughput sequencing and validated by Sanger sequencing. The genome is 34,080bp long and has 220 nucleotides long inverted terminal repeats. A total of 29 coding sequences were identified, 26 of which were functionally annotated. Among the unusual features of this genome is a remarkably long 4380bp E3 ORF1, that displays no sequence homology with the corresponding E3 regions of other adenoviruses. In addition, the fiber protein only has 26% identity with fiber proteins described in other adenoviruses. Three hypothetical proteins were predicted. The phylogenetic analysis indicates that the closest known relative to BdAdV-1 is an adenovirus detected in bottlenose dolphin (KR024710), with an amino acid sequence identity between 36 and 79% depending on the protein. Based on the phylogenic analysis, the BdAdV-1 appears to have co-evolved with its host. The results indicate that BdAdV-1 belongs to the Mastadenovirus genus of the Adenoviridae family, however, it is clearly different from other adenoviruses, especially in the 3'-end of the viral genome. The high degree of sequence divergence suggests that BdAdV-1 should be considered as a novel species in the Mastadenovirus genus. The study also demonstrates the usefulness of high-throughput sequencing to obtain full-length genomes of genetically divergent viruses., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Parainfluenza virus 3 (PIV-3) is a common viral infection not only in humans, but also in many other species. Serological evidence suggests that nearly 100 % of children in the United States have been infected with PIV-3 by 5 years of age. Similarly, in cattle, PIV-3 is commonly associated with bovine respiratory disease complex. A novel dolphin PIV-3 (TtPIV-1) was described by Nollens et al. in 2008 from a dolphin that was diagnosed with an unknown respiratory illness. At that time, TtPIV-1 was found to be most similar to, but distinct from, bovine PIV-3 (BPIV-3). In the present study, similar viral growth kinetics and pro-inflammatory cytokine (IL-1β, IL-6, and CXCL8) production were seen between BPIV-3 and TtPIV-1 in BEAS-2B, MDBK, and Vero cell lines. Initial nomenclature of TtPIV-1 was based on partial sequence of the fusion and RNA polymerase genes. Based on the similarities we saw with the in vitro work, it was important to examine the TtPIV-1 genome in more detail. Full genome sequencing and subsequent phylogenetic analysis revealed that all six viral genes of TtPIV-1 clustered within the recently described BPIV-3 genotype B strains, and it is proposed that TtPIV-1 be re-classified with BPIV-3 genotype B strains.

A northern Gulf of Mexico (GoM) cetacean unusual mortality event (UME) involving primarily bottlenose dolphins (Tursiops truncatus) in Louisiana, Mississippi, and Alabama began in February 2010 and continued into 2014. Overlapping in time and space with this UME was the Deepwater Horizon (DWH) oil spill, which was proposed as a contributing cause of adrenal disease, lung disease, and poor health in live dolphins examined during 2011 in Barataria Bay, Louisiana. To assess potential contributing factors and causes of deaths for stranded UME dolphins from June 2010 through December 2012, lung and adrenal gland tissues were histologically evaluated from 46 fresh dead non-perinatal carcasses that stranded in Louisiana (including 22 from Barataria Bay), Mississippi, and Alabama. UME dolphins were tested for evidence of biotoxicosis, morbillivirus infection, and brucellosis. Results were compared to up to 106 fresh dead stranded dolphins from outside the UME area or prior to the DWH spill. UME dolphins were more likely to have primary bacterial pneumonia (22% compared to 2% in non-UME dolphins, P = .003) and thin adrenal cortices (33% compared to 7% in non-UME dolphins, P = .003). In 70% of UME dolphins with primary bacterial pneumonia, the condition either caused or contributed significantly to death. Brucellosis and morbillivirus infections were detected in 7% and 11% of UME dolphins, respectively, and biotoxin levels were low or below the detection limit, indicating that these were not primary causes of the current UME. The rare, life-threatening, and chronic adrenal gland and lung diseases identified in stranded UME dolphins are consistent with exposure to petroleum compounds as seen in other mammals. Exposure of dolphins to elevated petroleum compounds present in coastal GoM waters during and after the DWH oil spill is proposed as a cause of adrenal and lung disease and as a contributor to increased dolphin deaths.

Background: Adenoviruses are common pathogens in vertebrates, including humans. In marine mammals, adenovirus has been associated with fatal hepatitis in sea lions. However, only in rare cases have adenoviruses been detected in cetaceans, where no clear correlation was found between presence of the virus and disease status., Case Presentation: A novel adenovirus was identified in four captive bottlenose dolphins with self-limiting gastroenteritis. Viral detection and identification were achieved by: PCR-amplification from fecal samples; sequencing of partial adenovirus polymerase (pol) and hexon genes; producing the virus in HeLa cells, with PCR and immunofluorescence detection, and with sequencing of the amplified pol and hexon gene fragments. A causative role of this adenovirus for gastroenteritis was suggested by: 1) we failed to identify other potential etiological agents; 2) the exclusive detection of this novel adenovirus and of seropositivity for canine adenoviruses 1 and 2 in the four sick dolphins, but not in 10 healthy individuals of the same captive population; and 3) the virus disappeared from feces after clinical signs receded. The partial sequences of the amplified fragments of the pol and hexon genes were closest to those of adenoviruses identified in sea lions with fatal adenoviral hepatitis, and to a Genbank-deposited sequence obtained from a harbour porpoise., Conclusion: These data suggest that adenovirus can cause self-limiting gastroenteritis in dolphins. This adenoviral infection can be detected by serology and by PCR detection in fecal material. Lack of signs of hepatitis in sick dolphins may reflect restricted tissue tropism or virulence of this adenovirus compared to those of the adenovirus identified in sea lions. Gene sequence-based phylogenetic analysis supports a common origin of adenoviruses that affect sea mammals. Our findings suggest the need for vigilance against adenoviruses in captive and wild dolphin populations.

Cetacean morbillivirus (CeMV) has caused several epizootics in multiple species of cetaceans globally and is an emerging disease among cetaceans in Australia. We detected CeMV in 2 stranded coastal Indo-Pacific bottlenose dolphins (Tursiops aduncus) in Western Australia. Preliminary phylogenetic data suggest that this virus variant is divergent from known strains.

A systemic morbillivirus infection was diagnosed postmortem in a juvenile bottlenose dolphin stranded in the eastern North Atlantic Ocean in 2005. Sequence analysis of a conserved fragment of the morbillivirus phosphoprotein gene indicated that the virus is closely related to dolphin morbillivirus recently reported in striped dolphins in the Mediterranean Sea.

While gammacoronaviruses mainly comprise infectious bronchitis virus (IBV) and its closely related bird coronaviruses (CoVs), the only mammalian gammacoronavirus was discovered from a white beluga whale (beluga whale CoV [BWCoV] SW1) in 2008. In this study, we discovered a novel gammacoronavirus from fecal samples from three Indo-Pacific bottlenose dolphins (Tursiops aduncus), which we named bottlenose dolphin CoV (BdCoV) HKU22. All the three BdCoV HKU22-positive samples were collected on the same date, suggesting a cluster of infection, with viral loads of 1 × 10(3) to 1 × 10(5) copies per ml. Clearance of virus was associated with a specific antibody response against the nucleocapsid of BdCoV HKU22. Complete genome sequencing and comparative genome analysis showed that BdCoV HKU22 and BWCoV SW1 have similar genome characteristics and structures. Their genome size is about 32,000 nucleotides, the largest among all CoVs, as a result of multiple unique open reading frames (NS5a, NS5b, NS5c, NS6, NS7, NS8, NS9, and NS10) between their membrane (M) and nucleocapsid (N) protein genes. Although comparative genome analysis showed that BdCoV HKU22 and BWCoV SW1 should belong to the same species, a major difference was observed in the proteins encoded by their spike (S) genes, which showed only 74.3 to 74.7% amino acid identities. The high ratios of the number of synonymous substitutions per synonymous site (Ks) to the number of nonsynonymous substitutions per nonsynonymous site (Ka) in multiple regions of the genome, especially the S gene (Ka/Ks ratio, 2.5), indicated that BdCoV HKU22 may be evolving rapidly, supporting a recent transmission event to the bottlenose dolphins. We propose a distinct species, Cetacean coronavirus, in Gammacoronavirus, to include BdCoV HKU22 and BWCoV SW1, whereas IBV and its closely related bird CoVs represent another species, Avian coronavirus, in Gammacoronavirus.

Morbilliviruses are recognized as biological agents highly impacting the health and conservation status of free-ranging cetaceans worldwide, as clearly exemplified by the two Dolphin Morbillivirus (DMV) epidemics of 1990-1992 and 2006-2008 among Mediterranean striped dolphins (Stenella coeruleoalba). After these two epidemics, morbilliviral infection (MI) cases with peculiar neurobiological features were reported in striped dolphins stranded along the Spanish coastline. Affected cetaceans showed a subacute-to-chronic, non-suppurative encephalitis, with brain lesions strongly resembling those found in human "subacute sclerosing panencephalitis" and "old dog encephalitis". Brain was the only tissue in which morbilliviral antigen and/or genome could be detected. Beside a case of morbilliviral encephalitis in a striped dolphin's calf stranded in 2009, we observed 5 additional MI cases in 2 striped dolphins, 1 bottlenose dolphin (Tursiops truncatus) and 2 fin whales (Balaenoptera physalus), all stranded in 2011 along the Italian coastline. Noteworthy, 3 of these animals (2 striped dolphins and 1 bottlenose dolphin) showed immunohistochemical (IHC) and/or biomolecular (PCR) evidence of morbilliviral antigen and/or genome exclusively in their brain, with 1 striped dolphin and 1 bottlenose dolphin also exhibiting a non-suppurative encephalitis. Furthermore, simultaneous IHC and PCR evidence of a Toxoplasma gondii coinfection was obtained in 1 fin whale. The above results are consistent with those reported in striped dolphins after the two MI epidemics of 1990-92 and 2006-2008, with evidence of morbilliviral antigen and/or genome being found exclusively in the brain tissue from affected animals., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The number of studies addressing neoplasia in marine mammals has recently increased, giving rise to concern whether such lesions could be reflective of an emerging infectious disease. Eight species-specific viruses, seven papillomaviruses (PVs) and two herpesviruses (HVs) have separately been shown to be associated with genital tumors in Atlantic bottlenose dolphins (Tursiops truncatus, Tt): TtPV1-6, as well as HVs provisionally assigned the names DeHV4 and -5 (Delphinid HVs). A definite causal role of these viruses in cell transformation remains to be demonstrated. Concurrent PV- and HV-infection has never been reported in marine mammals. DNA extractions from biopsies of genital tumors derived from 15 free-ranging Atlantic bottlenose dolphins were selected for molecular examination. Polymerase chain reaction (PCR) analyses revealed the presence of DeHV4, while a serological screening using an antibody-based TtPV enzyme-linked immunosorbent assay (ELISA) demonstrated previous and/or current infection of the HV-positive dolphins with at least one TtPV type. Therefore, care must be taken when drawing conclusions about viral causalities in tumor development, since the "hit and run" and other mechanisms have been described for types of both viral families. This study presents the first evidence of marine mammals having a history of PV- as well as HV-infection and discusses the disputed effects of viral co-infection., (Copyright © 2012 Elsevier B.V. All rights reserved.)

We report on the incidence of poxvirus-like lesions assessed by photographic identification in two estuarine populations of bottlenose dolphins (Tursiops aduncus) in Australia over a 3-year period. Poxvirus infections of odontocetes are characterized by pinhole or ring-like skin lesions that appear as solitary or coalesced circular gray blemishes. Environmental and physiological stressors are believed to contribute to their manifestation (Van Bressem et al., 2009b). A total of 187 boat-based surveys were completed from October 2003 to September 2006 in the Clarence River (CR) and Richmond River (RR) estuaries, with 720 dolphins sighted. Forty-six individuals, including calves, were identified in the CR and 23 in the RR. We investigated the temporal relationship between four flood events that occurred in the region during the study period and the occurrence of poxvirus-like skin lesions. Dolphin poxvirus-like lesions were not observed in these populations prior to 2004. Following flood events in 2004, 2005 and 2006, a total of 10 new cases were observed, 6 in the CR and 4 in the RR. Our data suggest that the occurrence of dolphin poxvirus-like lesions may be an indicator for climatic events such as flooding. Long-term follow-up of these estuarine populations is required to further clarify the factors leading to 'outbreaks' of poxvirus infections., (Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.)

We report serologic evidence of cetacean morbillivirus (CMV) infection in five of eight cetacean species found live stranded, injured, or trapped along the coast of southeastern Queensland and northern New South Wales, Australia between December 2005 and January 2011. Antibody to CMV was detected in 13 of 27 (48%) wild cetaceans sampled. Antibody prevalence was significantly higher in clinically diseased (69%) compared to nondiseased (18%) animals (P=0.018). There was high antibody prevalence (83%, n=6) in melon-headed whales (Peponocephala electra). Two of 13 (15%) captive cetaceans sampled between November 2005 and January 2011 had CMV antibodies and, as infection was unlikely to have occurred while in captivity, CMV infection appears to have been present in Australian wild cetaceans since at least 1985. These results indicate that morbillivirus infection is occurring without widespread cetacean mortality in this region. However, as the deaths of two immature Australian offshore bottlenose dolphins (Tursiops truncatus) were attributed to CMV infection, morbillivirus infection should be included in the differential diagnosis of disease in cetaceans in Australia. Captive cetacean populations may be prone to significant mortality as a result of CMV introduction, so strict quarantine procedures should be enforced when injured or stranded cetaceans are hospitalized and rehabilitated at Australian zoos and marine parks.

A juvenile offshore bottlenose dolphin (Tursiops truncatus) was found stranded with neurological signs and unable to swim or float unassisted. It subsequently died, succumbing to a combination of severe pneumonia and encephalitis. Morbillivirus serum neutralisation test serology was positive (titre 1:16) for cetacean morbillivirus and negative for both phocine distemper virus and canine distemper virus. There was concurrent thymic and lymph node lymphoid depletion and necrosis, together with intranuclear and intracytoplasmic acidophilic viral inclusion bodies and multinucleate syncytia within multiple organs. Paramyxovirus capsids were identified in lung sections via electron microscopy and morbillivirus antigen was demonstrated within sections of lung, thymus and brain by immunohistochemistry. Reverse transcription-polymerase chain reaction for morbillivirus nucleoprotein (N) and phosphoprotein (P) genes were positive and phylogenetic gene product sequence analysis revealed 98% and 94% sequence identity to dolphin morbillivirus, respectively. To the authors' knowledge, this is the first report of a cetacean mortality due to morbillivirus infection occurring in the southern hemisphere. Morbillivirus infection should be included in the differential diagnosis of stranded live or dead cetaceans in Australian waters, particularly if animals display neurological signs., (© 2011 The Authors. Australian Veterinary Journal © 2011 Australian Veterinary Association.)

Astroviruses are small, non-enveloped, positive-stranded RNA viruses. Previously studied mammalian astroviruses have been associated with diarrhoeal disease. Knowledge of astrovirus diversity is very limited, with only six officially recognized astrovirus species from mammalian hosts and, in addition, one human and some bat astroviruses were recently described. We used consensus PCR techniques for initial identification of five astroviruses of marine mammals: three from California sea lions (Zalophus californianus), one from a Steller sea lion (Eumetopias jubatus) and one from a bottlenose dolphin (Tursiops truncatus). Bayesian and maximum-likelihood phylogenetic analysis found that these viruses showed significant diversity at a level consistent with novel species. Astroviruses that we identified from marine mammals were found across the mamastrovirus tree and did not form a monophyletic group. Recombination analysis found that a recombination event may have occurred between a human and a California sea lion astrovirus, suggesting that both lineages may have been capable of infecting the same host at one point. The diversity found amongst marine mammal astroviruses and their similarity to terrestrial astroviruses suggests that the marine environment plays an important role in astrovirus ecology.

Genital epithelial tumors of Atlantic bottlenose dolphins (Tursiops truncatus [Tt]) and Burmeister's porpoises (Phocoena spinipinnis) were formerly shown to be associated with papillomavirus (PV) infection. Papillomaviruses are highly prevalent viruses involved in the development of various tumor types in a wide range of animals, and so-called high-risk PVs contribute to malignant progression. In marine mammals, the incidence and prevalence of PV infection, transmission pathways, and persistence of infection are largely unknown. Using virus-like particles of bottlenose dolphin PV type 1 (TtPV1) as the antigen, enzyme-linked immunosorbent assay (ELISA) studies were conducted to evaluate PV antibody prevalence in bottlenose dolphins. In total, sera obtained from 115 dolphins were examined. Fifty-one percent of captive dolphins (n=18 of 35) and 90% of free-ranging dolphins (n=72 of 80) were antibody positive. Higher ELISA reactivity was observed among males compared with females. Sexually immature dolphins appeared more likely to seroconvert with age. Besides determining their PV antibody prevalence, each animal was also assessed for the presence of orogenital tumors. Interestingly, the mean age of free-ranging dolphins with tumors (n=21) was 11.2 yr compared with 29.9 yr in captive dolphins with tumors (n=9). Results from the current study suggest PV infection in bottlenose dolphins is common, that the main route of PV transmission among them may be horizontal, and that orogenital neoplasia may develop in early life stages of certain free-ranging bottlenose dolphins.

An enterovirus was cultured from an erosive tongue lesion of a bottlenose dolphin (Tursiops truncatus). The morphology of virions on negative staining electron microscopy was consistent with those of enteroviruses. Analysis of 2613 bp of the polyprotein gene identified the isolate as a novel enterovirus strain, tentatively named bottlenose dolphin enterovirus (BDEV), that nests within the species Bovine enterovirus. Serologic evidence of exposure to enteroviruses was common in both free-ranging and managed collection dolphins. Managed collection dolphins were more likely to have high antibody levels, although the highest levels were reported in free-ranging populations. Associations between enterovirus antibody levels, and age, sex, complete blood counts, and clinical serum biochemistries were explored. Dolphins with higher antibody levels were more likely to be hyperproteinemic and hyperglobulinemic.

We studied the pathology, epidemiology, and clinical significance of genital herpesvirus infection in a zoo collection of bottlenose dolphins (Tursiops truncatus). Samples from the genital mucosa of male (n=21) and female (n=15) dolphins were tested by nested polymerase chain reaction (PCR) targeting the DNA polymerase of herpesvirus. Herpesvirus infection was significantly associated with the occurrence of mucosal plaques on penis (n=3) or vulva (n=4). Biopsies from a penile plaque showed epithelial hyperplasia by histology, contained herpesvirus-like particles by electron microscopy, and tested positive for herpesvirus by PCR. Herpesvirus was successfully cultivated from penile plaque samples and identified as a member of the Gammaherpesvirinae by DNA sequencing and phylogenetic analysis. We used the newly cultivated bottlenose dolphin herpesvirus (TTHV) to develop a direct enzyme-linked immunosorbent assay for anti-TTHV antibodies in banked sera of these dolphins. The percentage of positive samples was higher in adults (20/21, 95%) than in juveniles (7/15, 47%). Seroconversion occurred around the age of onset of sexual behavior. Although herpesvirus infection has been associated with abortion, perinatal mortality, and urogenital neoplasia in other species, we found no evidence of herpesvirus infection by PCR in tissues from six cases of abortion and perinatal mortality, and no diagnoses of urogenital tumors in 24 bottlenose dolphins from this zoo collection that died since 1990. Together, we here report the first successful cultivation from bottlenose dolphins of a herpesvirus that probably causes benign genital plaques, is endemic in this group of dolphins, and is likely transmitted by sexual contact.

Papillomaviruses (PVs) have been shown as being the etiologic agents of various benign and malignant tumours in many vertebrate species. In dolphins and porpoises, a high prevalence of orogenital tumours has recently been documented with at least four distinct novel species-specific PV types detected in such lesions. Therefore, we generated the immunological reagents to establish a serological screening test to determine the prevalence of PV infection in Atlantic bottlenose dolphins [(Tursiops truncatus (Tt)]. Using the baculovirus expression system, virus-like particles (VLPs) derived from the L1 proteins of two TtPV types, TtPV1 and TtPV2, were generated. Polyclonal antibodies against TtPV VLPs were produced in rabbits and their specificity for the VLPs was confirmed. Electron microscopy and enzyme-linked immunosorbent assay (ELISA) studies revealed that the generated VLPs self-assembled into particles presenting conformational immunodominant epitopes. As such, these particles are potential antigen candidates for a TtPV vaccine. Subsequently, the VLPs served as antigens in initial ELISA tests using sera from six bottlenose dolphins to investigate PV antibody presence. Three of these sera were derived from dolphins with genital tumour history and showed positive PV ELISA reactivity, while the remaining sera from lesion-free dolphins were PV antibody-negative. The results suggest that the developed screening test may serve as a potential tool for determining PV prevalence and thus for observing transmission rates in dolphin populations as the significance of PV infection in cetaceans starts to unfold.

A novel member of the parainfluenza virus family was identified in a bottlenose dolphin with respiratory disease. The case animal was a 19-year old male Atlantic bottlenose dolphin (Tursiops truncatus) that presented with signs of respiratory illness, including raspy, foul-odored breaths and cream-colored exudate from the blowhole. Focally extensive pyogranulomatous bronchointerstitial pneumonia with moderate numbers of intralesional yeast organisms was identified on histopathological examination. Other significant microscopic findings included multifocal erosive and ulcerative tracheitis and laryngitis consisting of active laryngeal lymphatic tissue and dilated glands with eosinophilic fluid. The cause of death was attributed to respiratory disease of unknown etiology. In addition to the postmortem isolation of Candida glabrata and mixed bacteria from lung tissue, a virus was isolated from two antemortem affected lung aspirates collected over a 2-month period and two postmortem samples (mediastinal lymph node and left lung tissue homogenate). The morphology of the virions on negative staining and transmission electron microscopy was consistent with that of paramyxoviruses. Two genomic fragments, comprising 532 and 419 nucleotides from the open reading frames that code for the viral polymerase and fusion protein, respectively, were amplified by polymerase chain reaction using degenerate primers. Phylogenetic analyses of the two viral RNA segments showed that the isolate comprised a novel virus strain, tentatively named T. truncatus parainfluenza virus type 1 (TtPIV-1). The virus is monophyletic with, but genetically distinct from, the various bovine parainfluenza virus type 3 strains.

Parainfluenza virus (PIV) is a leading cause of respiratory infections in humans. A novel virus closely related to human and bovine parainfluenza viruses types 3 (HPIV-3 and BPIV-3), named Tursiops truncatus parainfluenza virus type 1 (TtPIV-1), was isolated from a dolphin with respiratory disease. We developed a dolphin-specific ELISA to measure acute- and convalescent-phase PIV antibodies in dolphins during 1999-2006 with hemograms similar to that of the positive control. PIV seroconversion occurred concurrently with an abnormal hemogram in 22 animals, of which 7 (31.8%) had respiratory signs. Seroprevalence surveys were conducted on 114 healthy bottlenose dolphins in Florida and California. When the most conservative interpretation of positive was used, 11.4% of healthy dolphins were antibody positive, 29.8% were negative, and 58.8% were inconclusive. PIV appears to be a common marine mammal virus that may be of human health interest because of the similarity of TtPIV-1 to BPIV-3 and HPIV-3.

The immunization of exotic species presents considerable challenges. Nevertheless, for facilities like zoos, animal parks, government facilities and non-profit conservation groups, the protection of valuable and endangered species from infectious disease is a growing concern. The rationale for immunization in these species parallels that for human and companion animals; to decrease the incidence of disease. The U.S. Navy Marine Mammal Program, in collaboration with industry and academic partners, has developed and evaluated a DNA vaccine targeting a marine viral pathogen - dolphin morbillivirus (DMV). The DMV vaccine consists of the fusion (F) and hemagglutinin (H) genes of DMV. Vaccine constructs (pVR-DMV-F and pVR-DMV-H) were evaluated for expression in vitro and then for immunogenicity in mice. Injection protocols were designed for application in Atlantic bottlenose dolphins (Tursiops truncatus) to balance vaccine effectiveness with clinical utility. Six dolphins were inoculated, four animals received both pDMV-F and pDMV-H and two animals received a mock vaccine (vector alone). All animals received an inoculation week 0, followed by two booster injections weeks 8 and 14. Vaccine-specific immune responses were documented in all four vaccinated animals. To our knowledge, this is the first report of pathogen-specific immunogenicity to a DNA vaccine in an aquatic mammal species.

A novel papillomavirus (PV) was isolated from a genital condyloma of a free-ranging bottlenose dolphin inhabiting the coastal waters of Charleston Harbor, SC, USA: Tursiops truncatus papillomavirus type 2 (TtPV2). This novel virus represents the first isolated North American cetacean PV and the first American bottlenose dolphin PV. After the viral genome was cloned, sequenced and characterized genetically, phylogenetic analyses revealed that TtPV2 is most similar to the only published cetacean PV isolated and characterized thus far, Phocoena spinipinnis PV type 1 (PsPV1). A striking feature of the genome of TtPV2, as well as that of PsPV1, is the lack of an E7 open reading frame, which typically encodes one of the oncogenic proteins believed to be responsible for malignant transformation in the high-risk mucosotropic human papillomaviruses (HPVs). TtPV2 E6 contains a PDZ-binding motif that has been shown to be involved in transformation in the case of high-risk genital HPVs.

Herpesviruses and herpes-like viruses have been reported in only a small number of species of cetaceans, and, to date, clinical manifestations have been either as a life-threatening, disseminated infection or as a non-life-threatening dermatitis. A stranded juvenile Atlantic bottlenose dolphin, Tursiops truncatus, was admitted to the Dolphin and Whale Hospital for rehabilitation. On initial physical examination, the rostral skin had multifocal regions of hyperplasia, and the skin of the dorsum contained a large number of small papules. Histologically, epithelial hyperplasia was evident, and clusters of epithelial cells contained 5-15-microm intranuclear inclusion bodies. Transmission electron microscopic investigation revealed numerous 170-190-nm enveloped virions in both the intracellular spaces and the cytoplasm of epithelial cells, with numerous nucleocapsids noted in epithelial cell nuclei. Consensus primer polymerase chain reaction identified the presence of a novel herpesvirus associated with the lesions. Phylogenetic analysis of the deduced amino acid sequences of the herpesvirus DNA polymerase gene fragment showed it to align with alphaherpesvirus sequences from humans and domestic animals. Although clearly distinct, it was most closely related to two previously described alphaherpesviruses of dolphins. This case represents the first documentation of herpesvirus dermatitis in the Atlantic bottlenose dolphin.