Manatee calf call contour and acoustic structure varies by species and body size.

Vocal activity and signal characteristics of mammals are driven by several factors that result in both stability and plasticity over multiple time scales. All three extant species of manatee communicate with several calls that are especially important for maintaining contact between cows and calves. Determining if calf calls differ across manatee species will provide insights into the evolution of species-specific acoustic communication traits. We investigated the interspecific differences in the vocalizations of calves of Amazonian manatees (Trichechus inunguis) and the two subspecies of the West Indian manatee (T. manatus). Vocalizations of individual calves were recorded in rehabilitation centers in Brazil, Puerto Rico, the United States, and Mexico. The acoustic structure of calls produced by manatee calves varied between species and with body size. Amazonian manatee calves produced shorter calls with multiple notes at higher frequency while West Indian calves produced modulated calls that were lower in frequency and longer in duration. Smaller West Indian calves produced frequency modulated, hill-shaped calls that flattened with an increase in body length. Our results provide evidence for divergence in the ontogeny of vocalizations across T. manatus and T. inunguis and suggest variation in body size contributed to the evolution of differences in the characteristics of their calls.

Prevalence and genotype of Toxoplasma gondii in stranded Hawaiian cetaceans.

Toxoplasma gondii is a significant threat to endangered Hawaiian wildlife including birds and marine mammals. To estimate the prevalence of T. gondii in stranded cetaceans from 1997 to 2021 in Hawai'i, we tested tissues from 37 stranded spinner dolphins Stenella longirostris and 51 stranded individuals that represented 18 other cetacean species. DNA from cetacean tissue extracts were screened using a nested polymerase chain reaction (PCR) assay targeting the Toxoplasmatinae internal transcribed spacer 1 of the nuclear ribosomal DNA. A positive result was obtained in 9 tissues examined for each of 2 spinner dolphins out of 525 tissue samples analyzed by PCR. The PCR-positive spinner dolphins had disseminated acute toxoplasmosis with necrosis, inflammation, and intralesional protozoal cysts and tachyzoites in multiple organs. Discrete positive immunostaining for T. gondii was observed in all tissues tested including the adrenal gland, brain, liver, and lung. Both positive spinner dolphins were negative for cetacean morbillivirus. The T. gondii genotyping was performed by restriction fragment length polymorphism (PCR-RFLP) based on 10 genetic markers. The PCR-RFLP analysis revealed the T. gondii belonged to PCR-RFLP-ToxoDB genotype #24, previously detected in wild pig Sus scrofa in O'ahu, bobcats Lynx rufus from Mississippi, USA, and chickens Gallus gallus from Costa Rica and Brazil. These cases represent the first report of this genotype in aquatic mammals and the second and third reports of fatal disseminated T. gondii infection in stranded spinner dolphins from Hawai'i. Nearshore species, like spinner dolphins, may be at increased risk of mortality from this parasite in marine coastal waterways via sewage systems, storm water drainage, and freshwater runoff.

Characterization of novel papillomavirus from free-ranging Antillean manatee Trichechus manatus manatus with genital papillomatosis.

The Antillean manatee Trichechus manatus manatus is an Endangered species living along the Atlantic coasts of the Americas from Florida (USA), throughout the Caribbean, to Brazil. In July 2020, a manatee with multiple wounds due to boat-inflicted trauma was rescued from the coast east of Cayo Mata, Salinas, Puerto Rico. This manatee had neutropenia, leukopenia, and monocytosis associated with immunosuppression and nutritional deficiency anemia, as well as bacteria and fungi within the lesions. The manatee had genital lesions which included papules and linear plaques, microscopically characterized by mucosal hyperplasia with cytopathic changes typical of papillomavirus infection. Superficial epithelial cells had strong nuclear immunolabeling when examined using a monoclonal antibody specific to papillomavirus. The sequencing data of PCR products with papillomavirus-specific degenerative primers indicated that these lesions contained a novel manatee papillomavirus (Trichechus manatus papillomavirus, TmPV). The genomic DNA was amplified using a rolling circle amplification, and fully sequenced to be 7586 bp (GenBank accession no. OK073977). Other TmPVs were previously isolated from Florida manatees T. manatus latirostris. This novel virus was designated TmPV type 5 (TmPV5) based on its genomic characterization and sequence comparison. The TmPV5 genome shared 50.7, 48.9, 69.4, and 62.1% similarities with TmPV1, TmPV2, TmPV3, and TmPV4, respectively. TmPV5 is classified in the genus Rhopapillomavirus together with other manatee papillomaviruses. After 2.5 mo of veterinary treatment and rehabilitation, the manatee recovered and was released. This is the first report of papillomatosis in a free-ranging Antillean manatee.

Novel cetacean morbillivirus in a rare Fraser's dolphin (Lagenodelphis hosei) stranding from Maui, Hawai'i.

Cetacean morbillivirus (CeMV) is a global threat to cetaceans. We report a novel morbillivirus from a Fraser's dolphin (Lagenodelphis hosei) that stranded in Maui, Hawaii in 2018 that is dissimilar to the beaked whale morbillivirus previously identified from Hawaii and to other CeMV strains. Histopathological findings included intranuclear inclusions in bile duct epithelium, lymphoid depletion, rare syncytial cells and non-suppurative meningitis. Cerebellum and lung tissue homogenates were inoculated onto Vero.DogSLAMtag cells for virus isolation and cytopathic effects were observed, resulting in the formation of multinucleated giant cells (i.e., syncytia). Transmission electron microscopy of infected cell cultures also revealed syncytial cells with intracytoplasmic and intranuclear inclusions of viral nucleocapsids, consistent with the ultrastructure of a morbillivirus. Samples of the cerebellum, lung, liver, spleen and lymph nodes were positive for morbillivirus using a reverse transcription-polymerase chain reaction. The resulting 559 bp L gene sequence had the highest nucleotide identity (77.3%) to porpoise morbillivirus from Northern Ireland and the Netherlands. The resulting 248 bp P gene had the highest nucleotide identity to porpoise morbillivirus in Northern Ireland and the Netherlands and to a stranded Guiana dolphin (Sotalia guianensis) in Brazil (66.9%). As Fraser's dolphins are a pelagic species that infrequently strand, a novel strain of CeMV may be circulating in the central Pacific that could have additional population impacts through transmission to other small island-associated cetacean species.

Genomic characterization of a novel circovirus from a stranded Longman's beaked whale (Indopacetus pacificus).

Tissues from a juvenile Longman's beaked whale that stranded in Hawaii in 2010 were screened for viruses using a Next-Generation Sequencing (NGS) approach. From the NGS data, the full genome (1,849 bp) of a novel beaked whale circovirus (BWCV) was determined. Two open reading frames (ORF) were annotated, including ORF1 that encodes the capsid gene, ORF2 that encodes the replication-associated gene, and a 9-bp conserved nonamer on the apex of the open loop found in all circoviruses. Independent phylogenetic analyses based on amino acid sequence alignments of the two CV proteins supported the BWCV as a member of the genus Circovirus, branching as the sister species to the recently discovered canine circovirus. A sequence identity matrix generated from complete genome alignments revealed the BWCV displays between from 51.1 to 56.7% nucleotide identity to other circoviruses, which is lower than the 80% threshold proposed for species demarcation. Considering the genetic and phylogenetic analyses, we propose the formal species designation of beaked whale circovirus. An endpoint PCR assay targeting the BWCV genome confirmed the presence of the BWCV DNA in every tissue from which DNA was extracted, including spleen, muscle, left ventricle, left adrenal gland, liver, lung, cerebrum, cerebellum, and lymph node. An automated in situ hybridization assay utilizing RNAscope® technology and targeting the replication-associated gene resulted in labeling of individual cells morphologically resembling mononuclear leukocytes and cells of blood vessels in diaphragm, liver, lymph nodes, lung, pericardium, oral mucosa and tongue, adrenal gland, testis, aorta, intestine, stomach and heart. The clinical or pathologic significance of BWCV is undetermined, as are its host range, prevalence, and pathogenicity in cetaceans of Hawaiian waters and elsewhere.

Genome Sequence of a Gammaherpesvirus from a Common Bottlenose Dolphin (Tursiops truncatus).

A herpesvirus genome was sequenced directly from a biopsy specimen of a rectal lesion from a female common bottlenose dolphin. This genome sequence comprises a unique region (161,235 bp) flanked by multiple copies of a terminal repeat (4,431 bp) and contains 72 putative genes. The virus was named common bottlenose dolphin gammaherpesvirus 1.

Ranavirus phylogenomics: Signatures of recombination and inversions among bullfrog ranaculture isolates.

Ranaviruses are emerging pathogens of fish, amphibians, and reptiles that threaten aquatic animal industries and wildlife worldwide. Our objective was to genetically characterize ranaviruses isolated during separate bullfrog Lithobates catesbeianus die-offs that occurred eight years apart on the same North American farm. The earlier outbreak was due to a highly pathogenic strain of common midwife toad virus (CMTV) previously known only from Europe and China. The later outbreak was due to a chimeric ranavirus that displayed a novel genome arrangement and a DNA backbone typical for Frog virus 3 (FV3) strains except for interspersed fragments acquired through recombination with the CMTV isolated earlier. Both bullfrog ranaviruses are more pathogenic than wild-type FV3 suggesting recombination may have resulted in the increased pathogenicity observed in the ranavirus isolated in the later outbreak. Our study underscores the role international trade in farmed bullfrogs may have played in the global dissemination of highly pathogenic ranaviruses.

Coinfection by Cetacean morbillivirus and Aspergillus fumigatus in a juvenile bottlenose dolphin (Tursiops truncatus) in the Gulf of Mexico.

A recently deceased juvenile male bottlenose dolphin (Tursiops truncatus) was found floating in the Gulf of Mexico, off Sand Key in Clearwater, Florida. At autopsy, we identified pneumonia and a focus of malacia in the right cerebrum. Cytologic evaluation of tissue imprints from the right cerebrum revealed fungal hyphae. Fungal cultures of the lung and brain yielded Aspergillus fumigatus, which was confirmed by amplification of a portion of the fungal nuclear ribosomal internal transcribed spacer 2 region sequence. Microscopic pulmonary lesions of bronchiolar epithelial cell syncytia with intracytoplasmic and intranuclear inclusions within bronchiolar epithelial cells were suggestive of Cetacean morbillivirus (CeMV) infection. The occurrence of CeMV infection was supported by positive immunohistochemical staining for morbillivirus antigen. CeMV detection was confirmed by amplification and sequencing a portion of the morbilliviral RNA-dependent RNA polymerase gene from lung tissue. This case provides CeMV sequence data available from the Gulf of Mexico and underscores the need for genomic sequencing across diverse host, temporospatial, and population (i.e., single animal vs. mass mortality events) scales to improve our understanding of these globally emerging pathogens.

Acoustical and anatomical determination of sound production and transmission in West Indian (Trichechus manatus) and Amazonian (T. inunguis) manatees.

West Indian (Trichechus manatus) and Amazonian (T. inunguis) manatees are vocal mammals, with most sounds produced for communication between mothers and calves. While their hearing and vocalizations have been well studied, the actual mechanism of sound production is unknown. Acoustical recordings and anatomical examination were used to determine the source of sound generation. Recordings were performed on live captive manatees from Puerto Rico, Cuba and Colombia (T. manatus) and from Peru (T. inunguis) to determine focal points of sound production. The manatees were recorded using two directional hydrophones placed on the throat and nasal region and an Edirol-R44 digital recorder. The average sound intensity level was analyzed to evaluate the sound source with a T test: paired two sample for means. Anatomical examinations were conducted on six T. manatus carcasses from Florida and Puerto Rico. During necropsies, the larynx, trachea, and nasal areas were dissected, with particular focus on identifying musculature and soft tissues capable of vibrating or constricting the airway. From the recordings we found that the acoustical intensity was significant (P < 0.0001) for both the individuals and the pooled manatees in the ventral throat region compared to the nasal region. From the dissection we found two raised areas of tissue in the lateral walls of the manatee's laryngeal lumen that are consistent with mammalian vocal folds. They oppose each other and may be able to regulate airflow between them when they are adducted or abducted by muscular control of arytenoid cartilages. Acoustic and anatomical evidence taken together suggest vocal folds as the mechanism for sound production in manatees.