Unusual striped dolphin mass mortality episode related to cetacean morbillivirus in the Spanish Mediterranean sea.

BACKGROUND: In the last 20 years, Cetacean Morbillivirus (CeMV) has been responsible for many die-offs in marine mammals worldwide, as clearly exemplified by the two dolphin morbillivirus (DMV) epizootics of 1990-1992 and 2006-2008, which affected Mediterranean striped dolphins (Stenella coeruleoalba). Between March and April 2011, the number of strandings on the Valencian Community coast (E Spain) increased. CASE PRESENTATION: Necropsy and sample collection were performed in all stranded animals, with good state of conservation. Subsequently, histopathology, immunohistochemistry, conventional reverse transcription polymerase chain reaction (RT-PCR) and Universal Probe Library (UPL) RT-PCR assays were performed to identify Morbillivirus. Gross and microscopic findings compatible with CeMV were found in the majority of analyzed animals. Immunopositivity in the brain and UPL RT-PCR positivity in seven of the nine analyzed animals in at least two tissues confirmed CeMV systemic infection. Phylogenetic analysis, based on sequencing part of the phosphoprotein gene, showed that this isolate is a closely related dolphin morbillivirus (DMV) to that responsible for the 2006-2008 epizootics. CONCLUSION: The combination of gross and histopathologic findings compatible with DMV with immunopositivity and molecular detection of DMV suggests that this DMV strain could cause this die-off event.

Molecular diagnosis of lobomycosis-like disease in a bottlenose dolphin in captivity.

We report the diagnosis and molecular characterization of lobomycosis-like lesions in a captive bottlenose dolphin. The clinical picture and the absence of growth in conventional media resembled the features associated with Lacazia loboi. However sequencing of ribosomal DNA and further phylogenetic analyses showed a novel sequence more related to Paracoccidioides brasilensis than to L. loboi. Moreover, the morphology of the yeast cells differed from those L. loboi causing infections humans. These facts suggest that the dolphin lobomycosis-like lesions might have been be caused by different a different fungus clustered inside the order Onygenales. A successful treatment protocol based on topic and systemic terbinafine is also detailed.

Diagnosis of Cetacean morbillivirus: A sensitive one step real time RT fast-PCR method based on SYBR(®) Green.

Cetacean morbillivirus (CeMV) (family Paramyxoviridae, genus Morbillivirus) is considered the most pathogenic virus of cetaceans. It was first implicated in the bottlenose dolphin (Tursiops truncatus) mass stranding episode along the Northwestern Atlantic coast in the late 1980s, and in several more recent worldwide epizootics in different Odontoceti species. This study describes a new one step real-time reverse transcription fast polymerase chain reaction (real-time RT-fast PCR) method based on SYBR(®) Green to detect a fragment of the CeMV fusion protein gene. This primer set also works for conventional RT-PCR diagnosis. This method detected and identified all three well-characterized strains of CeMV: porpoise morbillivirus (PMV), dolphin morbillivirus (DMV) and pilot whale morbillivirus (PWMV). Relative sensitivity was measured by comparing the results obtained from 10-fold dilution series of PMV and DMV positive controls and a PWMV field sample, to those obtained by the previously described conventional phosphoprotein gene based RT-PCR method. Both the conventional and real-time RT-PCR methods involving the fusion protein gene were 100- to 1000-fold more sensitive than the previously described conventional RT-PCR method.

Simultaneous diagnosis of Cetacean morbillivirus infection in dolphins stranded in the Spanish Mediterranean sea in 2011 using a novel Universal Probe Library (UPL) RT-PCR assay.

A highly sensitive and specific real-time (rt) RT-PCR assay has been developed for rapid, simultaneous detection of three strains of cetacean morbillivirus (CeMV). In this assay, two PCR primers and a hydrolysis probe from a commercially available Universal Probe Library (UPL) are used to amplify a highly conserved region within the fusion protein gene. RT-PCR is carried out on the same sample using two primer sets in parallel: one set detects the more virulent strains, dolphin morbillivirus (DMV) and porpoise morbillivirus (PMV), and the other set detects the least virulent and least common strain, pilot whale morbillivirus (PWMV). Sensitivity analysis using dilute samples containing purified DMV, PMV and PWMV showed that viral RNA detection limits in this UPL RT-PCR assay were lower than in a conventional RT-PCR assay. Our method gave no amplification signal with field samples positive for viruses related and unrelated to CeMV, such as phocine distemper virus (PDV). The reliability and robustness of the UPL RT-PCR assay were verified using tissue samples previously analyzed by conventional methods, as well as a panel of clinical samples suspected of containing CeMV. Using the UPL RT-PCR assay, we were able to associate DMV with a mass stranding of striped dolphins in the Spanish Mediterranean in 2011 with greater reliability than was possible with a conventional RT-PCR method. These results suggest that this UPL RT-PCR method is more sensitive and specific than the conventional approach, and that it may be an affordable and rapid test for routine diagnosis of three CeMV strains.

Genetic comparison among dolphin morbillivirus in the 1990-1992 and 2006-2008 Mediterranean outbreaks.

In 1990, dolphin morbillivirus (DMV) killed thousands of striped dolphins in the Mediterranean. Subsequently, the prevalence of the infection declined in this species. In 2006-2008, the virus killed not only numerous striped dolphins but also long-finned pilot whales. All partial sequences of the phosphoprotein and nucleoprotein genes obtained thus far from different host species during the 2006-2008 outbreak show 100% identity, suggesting that a single virus was involved, and these sequences are nearly identical to the 1990 Spanish strain. Here our first objective was to determine the sequence identity between the morbillivirus from the 2006-2008 outbreak and the 1990 Spanish strain by sequencing more extensive genomic regions of strains from one pilot whale and one striped dolphin stranded in 2007. The second objective was to investigate the relationship between the 1990 and 2007 strains by constructing a phylogenetic tree based on the phosphoprotein gene to compare several Cetacean morbilliviruses, and another tree based on the nearly complete genomes of Mediterranean DMV. The third objective was to identify the most variable regions in the DMV genomes. Results showed that the two 2007 Spanish strains were 99.9% identical over 9050 bp and should be considered the same virus, and that this virus is 99.3-99.4% similar to the 1990 Spanish strain. The phylogenetic trees, together with the common geographical area for the two outbreaks, suggest that the 2007 DMV strains evolved from the 1990 DMV strain. Pilot whales do not seem to have been exposed or infected during the 1990-1992 epidemic, since these populations appeared to be immunologically naïve in 2006-2008. Our results suggest that the virus may have evolved in striped dolphin populations prior to the 2006-2008 outbreak, after which it entered the long-finned pilot whale, perhaps aided by an alanine to valine mutation in the N-terminal domain of the fusion protein.