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.

Medical management and resolution of perinatal bilateral exophthalmia and secondary corneal ulcers in an Antillean manatee (Trichechus manatus manatus) neonate from Puerto Rico.

OBJECTIVE: To present a novel case of perinatal bilateral exophthalmos and corneal ulcers in a neonate Antillean manatee and describe the medical treatment that led to the resolution of the observed clinical signs and vision restoration. ANIMAL STUDIED: A manatee stranded alone in Puerto Rico on July 5, 2020. RESULTS: The manatee was found in critical condition with pronounced exophthalmos, lagophthalmos, and corneal opacification of both eyes (OU). Vision impairment was evident due to the lack of ocular menace reflex and bumping into the tank's walls. Biomicroscopy revealed conjunctival hyperemia and chemosis, limited third eyelid movement, but had viscous tears present OU. Dense, full-thickness, white to cream-colored cellular infiltrates affected 70% of the cornea with peripheral active vascularization OU. Rubeosis iridis was also present OU. Treatment consisted of supportive medical management, including nutritional support and topical treatment for ulcerative keratitis. Resolution of the corneal ulcers and functional vision were achieved after 6 weeks of therapy. Currently, bilateral, mild, intermittent exophthalmos is observed with no adverse clinical signs, and the calf is in good health. CONCLUSIONS: The extent of bilateral corneal disease on a neonatal calf may be a result of an intrauterine infection or possible trauma at or right after birth. While the latter may have led to exophthalmia and consequent corneal disease, the exact cause could not be determined. Supportive therapy and medical management of infectious keratitis were successful and led to vision recovery. This is the first report of ocular pathology in a neonatal manatee.

Baseline urinalysis results in 32 healthy Antillean manatees (Trichechus manatus manatus).

OBJECTIVE: To describe results of analysis of free-catch urine samples collected from Antillean manatees (Trichechus manatus manatus) under human care in the Caribbean. ANIMALS: 32 Antillean manatees in 5 Caribbean oceanaria and rescue centers. PROCEDURES: Urine samples were obtained by opportunistic free catch during physical examination or through the use of operant conditioning procedures. Urinalyses consisted of macro- and microscopic evaluations, biochemical analyses with test strips, and refractometry. Results were compared for manatees grouped on the basis of age, sex, and habitat. RESULTS: Urine samples were typically clear, straw colored, and alkaline (mean pH, 8.0); had a urinoid odor and low specific gravity (mean, 1.010); and had results on qualitative test strips that were consistently negative for the presence of glucose, bilirubin, ketones, proteins, nitrites, RBCs, and WBCs. Microscopically, the mean ± SD number of RBCs and WBCs/hpf was 0.5 ± 0.3 RBCs/hpf and 1.1 ± 1.5 WBCs/hpf. The presence of some epithelial cells and crystals was typical. Spermatozoa were found in urine from 1 of 15 sexually mature males, and parasite larvae and eggs were found in urine from 2 manatees. CONCLUSIONS AND CLINICAL RELEVANCE: Results of the present study yielded the first compilation of baseline urinalysis values in healthy Antillean manatees under human care, which, when combined with physical examination and other diagnostic procedures, can help in monitoring the health of these animals. We encourage the use of free-catch urine collection methods, as used in the present study, for routine urinalyses of manatees under human care in zoos, aquaria, or rescue centers.

Manual Collection and Semen Characterization in a West Indian Manatee (Trichechus manatus).

Limited information is available regarding male reproductive physiology in West Indian manatees (Trichechus manatus). Currently, only basic ultrastructural and morphometric descriptions of the spermatozoon exist; however, there are no reports evaluating any seminal characteristics in this species. Therefore, the aim of the study was to fill current gaps in knowledge regarding semen parameters in West Indian manatees by collecting and characterizing multiple ejaculate samples from a single, adult West Indian manatee. Samples were analyzed for the following semen parameters: volume, agglutination, pH, osmolality, viscosity, concentration, total sperm number, motility and kinematic parameters, morphology, plasma membrane integrity, acrosome integrity, chromatin maturation, and chromatin condensation. All macroscopic semen parameters varied to some extent between samples. Total and progressive motility was high for ejaculates 2 to 5, exceeding 97 and 89%, respectively; however, these parameters decreased dramatically throughout ejaculates 6 and 7. Across all samples, curvilinear velocity, straight-line velocity, and average pathway velocity represented the largest significant differences (p < 0.001) between each of the progression velocity subgroups (rapid, medium, slow). Sperm characteristics, including acrosome integrity (79.8%), chromatin condensation (93.1%), and chromatin maturation (99.5%) were very high; however, high numbers of morphologically abnormal sperm were present (52.9%) and plasma membrane integrity was low (45.1%). These results are the first of their kind for this species and suggest high semen quality, based on multiple ejaculates, in this male West Indian manatee.

Baseline urinalysis values in common bottlenose dolphins under human care in the Caribbean.

Urinalysis is a rapid, simple, inexpensive, and reliable test that documents urine abnormalities reflecting various types of renal, hormonal, or metabolic diseases. Urinalysis could assist proper monitoring of the health of dolphins under human care; however, normal baseline values for dolphin urinalysis have not been reported, to our knowledge. We sampled urine from 193 common bottlenose dolphins ( Tursiops truncatus), living under human care in 24 Caribbean dolphinariums, by voluntary free-catch and analyzed the urine for chemical and microscopic variables using multi-agent dry reagent chemistry dipstick test strips, dedicated pH reagent strips, and unstained sediment slides. Most urine was clear, pale yellow to dark yellow, and had a fishy odor. Dipstick glucose, bilirubin, ketones, and nitrites were negative in all dolphins. The urine pH was acidic ( x¯ ± SD; 5.88 ± 0.58) and specific gravity (SG) was 1.035 ± 0.008. Most animals had 0-2 red blood cells and white blood cells per 40× field, and were negative for proteins. On microscopic sediment, 42.7% of samples had few-to-many squamous epithelial cells; hyaline and epithelial casts were observed only rarely. Crystals were observed in 36.6% of the samples; most were calcium oxalate dihydrate (48.2%) and amorphous urates (42.4%). The values obtained in our study can be used as a reference for health monitoring of dolphins in dolphinariums, and to monitor renal conditions and function in dolphins being rehabilitated or under human care.

Disseminated toxoplasmosis in Antillean manatees Trichechus manatus manatus from Puerto Rico.

Necropsies were conducted on 4 Antillean manatees Trichechus manatus manatus that were stranded in single events on the coastal beaches of Puerto Rico from August 2010 to August 2011. Three manatees were emaciated and the gastrointestinal tracts were devoid of digesta. Microscopically, all manatees had severe widespread inflammatory lesions of the gastrointestinal tract and heart with intralesional tachyzoites consistent with Toxoplasma gondii identified by histological, ultrastructural and immunohistochemical techniques. The gastrointestinal lesions included severe, multifocal to diffuse, chronic-active enteritis, colitis and/or gastritis often with associated ulceration, necrosis and hemorrhage. Enteric leiomyositis was severe and locally extensive in all cases and associated with the most frequently observed intralesional protozoans. Moderate to severe, multifocal, chronic to chronic-active, necrotizing myocarditis was also present in all cases. Additionally, less consistent inflammatory lesions occurred in the liver, lung and a mesenteric lymph node and were associated with fewer tachyzoites. Sera (n = 30) collected from free-ranging and captive Puerto Rican manatees and a rehabilitated/released Puerto Rican manatee from 2003 to 2012 were tested for antibodies for T. gondii. A positive T. gondii antibody titer was found in 2004 in 1 (3%) of the free-ranging cases tested. Disease caused by T. gondii is rare in manatees. This is the first report of toxoplasmosis in Antillean manatees from Puerto Rico. Additionally, these are the first reported cases of disseminated toxoplasmosis in any sirenian. The documentation of 4 cases of toxoplasmosis within one year and the extremely low seroprevalence to T. gondii suggest that toxoplasmosis may be an emerging disease in Antillean manatees from Puerto Rico.