Otariid gammaherpesvirus 1 in South American fur seals (Arctocephalus australis) and a novel related herpesvirus in free-ranging South American sea lions (Otaria byronia): Prevalence and effects of age, sex, and sample type.

Otariid gammaherpesvirus 1 (OtGHV1) is associated with high rates of urogenital carcinoma in free-ranging California sea lions (Zalophus californianus; CSL), and until recently was reported only in the Northern Hemisphere. The objective of this study was to survey free-ranging South American sea lions (Otaria byronia; SASL) and South American fur seals (Arctocephalus australis: SAFS) in Punta San Juan, Peru for OtGHV1 and to determine prevalence characteristics. Twenty-one percent (14/67) of urogenital swabs collected over three years (2011, 2014, 2015) from live pinnipeds of both species tested positive with a pan-herpesvirus conventional PCR. Sequencing of SAFS amplicons revealed 100% homology to OtGHV1 at the DNA polymerase, glycoprotein B, and viral bcl2-like genes. Sequencing of SASL amplicons revealed a novel related virus, herein called Otariid gammaherpesvirus 8 (OtGHV8). For comparison of sample sites, urogenital, conjunctival, and oropharyngeal swabs collected from 136 live pinnipeds of both species at Punta San Juan between 2011-2018 were then assayed using quantitative PCR for a segment of the OtGHV1/8 DNA polymerase gene using a qPCR assay now determined to cross-react between the two viruses. In total, across both species, 38.6% (51/132) of urogenital swabs, 5.6% (4/71) of conjunctival swabs, and 1.1% (1/90) of oropharyngeal swabs were positive for OtGHV1/8, with SASL only positive on urogenital swabs. Results from SASL were complicated by the finding of OtGHV8, necessitating further study to determine prevalence of OtGHV1 versus OtGHV8 using an alternate assay. Results from SAFS suggest a potential relationship between OtGHV1 in SAFS and CSL. Though necropsy surveillance in SAFS is very limited, geographic patterns of OtGHV1-associated urogenital carcinoma in CSL and the tendency of herpesviruses to cause more detrimental disease in aberrant hosts suggests that it is possible that SAFS may be the definitive host of OtGHV1, which gives further insight into the diversity and phyogeography of this clade of related gammaherpesviruses.

Environmental Enrichment Devices Are Safe and Effective at Reducing Undesirable Behaviors in California Sea Lions and Northern Elephant Seals during Rehabilitation.

Environmental enrichment devices (EEDs) have been proven to promote positive wellbeing in zoos and aquariums, and support animals' reintroduction success; however, their use in rehabilitation centers is still limited. This pilot study investigated the safety and efficacy of three EEDs, Artificial Kelp, Horse KONG™ and Wubba Kong™, and their ability to decrease and/or eliminate undesired stereotypic behaviors or looking at staff/staff areas in seven wild California sea lions (CSLs) and eight northern elephant seals (NESs) undergoing rehabilitation in Southern California. Observers conducted instantaneous sampling once a minute during a 30-min baseline, followed by a 30-min EED implementation on one focal animal at a time. The data were analyzed with generalized linear mixed models (GLMM). All three EEDs were found to be durable and safe throughout the study. Our results show a significant reduction in stereotypical behaviors compared to no EED treatments in CSLs, with the most significant effect being with the Horse KONG™. The Wubba KONG™ and Artificial Kelp provisions also reduced the undesired behavior in both species of being focused on human caretakers. Individual preferences for specific EEDs were found within species and between species, with the NESs using EEDs more than the CSLs. This study highlights the beneficial aspect of EEDs for pinnipeds in rehabilitation centers for improving their quality of life.

Draft Genome Sequences of Erysipelothrix sp. Strains Isolated from Stranded Septic Bottlenose Dolphins in Alabama, USA.

We report the genome sequences of two Erysipelothrix isolates from fatal cases of sepsis in bottlenose dolphins (Tursiops truncatus). The genomes were found to be most closely related to Erysipelothrix rhusiopathiae and Erysipelothrix piscisicarius. This information expands our knowledge of the genetic characteristics of this pathogen, which can affect free-ranging marine mammals.

Cardiac assessments of bottlenose dolphins (Tursiops truncatus) in the Northern Gulf of Mexico following exposure to Deepwater Horizon oil.

The Deepwater Horizon (DWH) oil spill profoundly impacted the health of bottlenose dolphins (Tursiops truncatus) in Barataria Bay, LA (BB). To comprehensively assess the cardiac health of dolphins living within the DWH oil spill footprint, techniques for in-water cardiac evaluation were refined with dolphins cared for by the U.S. Navy Marine Mammal Program in 2018 and applied to free-ranging bottlenose dolphins in BB (n = 34) and Sarasota Bay, Florida (SB) (n = 19), a non-oiled reference population. Cardiac auscultation detected systolic murmurs in the majority of dolphins from both sites (88% BB, 89% SB) and echocardiography showed most of the murmurs were innocent flow murmurs attributed to elevated blood flow velocity [1]. Telemetric six-lead electrocardiography detected arrhythmias in BB dolphins (43%) and SB dolphins (31%), all of which were considered low to moderate risk for adverse cardiac events. Echocardiography showed BB dolphins had thinner left ventricular walls, with significant differences in intraventricular septum thickness at the end of diastole (p = 0.002), and left ventricular posterior wall thickness at the end of diastole (p = 0.033). BB dolphins also had smaller left atrial size (p = 0.004), higher prevalence of tricuspid valve prolapse (p = 0.003), higher prevalence of tricuspid valve thickening (p = 0.033), and higher prevalence of aortic valve thickening (p = 0.008). Two dolphins in BB were diagnosed with pulmonary arterial hypertension based on Doppler echocardiography-derived estimates and supporting echocardiographic findings. Histopathology of dolphins who stranded within the DWH oil spill footprint showed a significantly higher prevalence of myocardial fibrosis (p = 0.003), regardless of age, compared to dolphins outside the oil spill footprint. In conclusion, there were substantial cardiac abnormalities identified in BB dolphins which may be related to DWH oil exposure, however, future work is needed to rule out other hypotheses and further elucidate the connection between oil exposure, pulmonary disease, and the observed cardiac abnormalities.

Unlocking the Role of a Genital Herpesvirus, Otarine Herpesvirus 1, in California Sea Lion Cervical Cancer.

Urogenital carcinoma in California sea lions (Zalophus californianus) is the most common cancer of marine mammals. Primary tumors occur in the cervix, vagina, penis, or prepuce and aggressively metastasize resulting in death. This cancer has been strongly associated with a sexually transmitted herpesvirus, otarine herpesvirus 1 (OtHV1), but the virus has been detected in genital tracts of sea lions without cancer and a causative link has not been established. To determine if OtHV1 has a role in causing urogenital carcinoma we sequenced the viral genome, quantified viral load from cervical tissue from sea lions with (n = 95) and without (n = 163) urogenital carcinoma, and measured viral mRNA expression using in situ mRNA hybridization (Basescope®) to quantify and identify the location of OtHV1 mRNA expression. Of the 95 sea lions diagnosed with urogenital carcinoma, 100% were qPCR positive for OtHV1, and 36% of the sea lions with a normal cervix were positive for the virus. The non-cancer OtHV1 positive cases had significantly lower viral loads in their cervix compared to the cervices from sea lions with urogenital carcinoma. The OtHV1 genome had several genes similar to the known oncogenes, and RNA in situ hybridization demonstrated high OtHV1 mRNA expression within the carcinoma lesions but not in normal cervical epithelium. The high viral loads, high mRNA expression of OtHV1 in the cervical tumors, and the presence of suspected OtHV1 oncogenes support the hypothesis that OtHV1 plays a significant role in the development of sea lion urogenital carcinoma.

Health Impacts and Recovery From Prolonged Freshwater Exposure in a Common Bottlenose Dolphin (Tursiops truncatus).

Common bottlenose dolphins (Tursiops truncatus) exposed to freshwater or low salinity (<10 practical salinity units; PSU) for prolonged periods of time have been documented to develop skin lesions, corneal edema and electrolyte abnormalities, and in some instances they have died. Here we review a case of an out-of-habitat subadult, female common bottlenose dolphin that remained in a freshwater lake in Seminole, Alabama for at least 32 days. Due to concerns for the dolphin's health a rescue was initiated. At the time of rescue bloodwork results indicated minor electrolyte abnormalities (hyponatremia, hypochloremia, hypoosmolality). Renal function was not affected (normal creatinine and urea nitrogen) and all other bloodwork parameters (hemogram; serum biochemistry analytes) were within normal limits. The dolphin was deemed healthy enough for immediate relocation and release. A satellite-linked tag was attached to the dorsal fin to track the dolphin following its relocation to a nearby brackish water bay (Perdido Bay, AL), a known habitat for bottlenose dolphins. Twelve weeks following release, the dolphin was found dead as a result of a fisheries interaction (peracute underwater entrapment). A full necropsy was conducted and there was complete resolution of the skin pallor and skin lesions and no evidence of chronic renal or central nervous system lesions. Post-mortem analysis of vitreous humor (used as a proxy for serum analytes and to determine post-mortem interval) was challenging to interpret and has not been validated in dolphins. This supports the need for future research in cetaceans to establish a species-specific approach. Elevated barium (Ba) concentrations in tooth dentin corresponded to increased seasonal freshwater discharge patterns, confirming repeated annual exposure to low salinity conditions prior to death and indicating freshwater exposure may pose an ongoing threat to dolphins in the region. This case provides a unique opportunity to follow the progression of prolonged freshwater exposure and recovery in a bottlenose dolphin and highlights that dolphins in nearshore habitats face a combination of persistent natural and human associated threats.

Pregnancy profiles in the common bottlenose dolphin (Tursiops truncatus): Clinical biochemical and hematological variations during healthy gestation and a successful outcome.

The physiological demands of pregnancy inevitably result in changes of both biochemical and hematological parameters as the fetus develops. Alterations in blood parameters have been observed to shift according to both trimester and species, to support fetal physiological needs and maternal basal requirements. Establishing normal reference ranges for each stage in gestation is important to facilitate diagnosis of underlying health concerns and prevent over-diagnosing abnormalities. Despite bottlenose dolphins (Tursiops truncatus) being one of the most highly studied cetaceans, the blood profile changes occurring as a result of pregnancy have not been previously described. A retrospective analysis was performed from blood samples obtained from 42 successful pregnancies from 20 bottlenose dolphins in a managed population over 30 years. Samples were compared to non-pregnant states and among trimesters of pregnancy. Blood profile fluctuations occurred throughout gestation, however significant alterations predominantly occurred between the 2nd and 3rd trimester. Hematological changes from the 2nd to the 3rd trimester included a decrease in lymphocytes, decrease in platelet count, and hemoconcentration with increased hematocrit and hemoglobin. Biochemical changes in the 3rd trimester included significant reductions in ALKP (alkaline phosphatase), ALT (alanine aminotransferase) and AST (aspartate aminotransferase) with significant increases observed in albumin, globulins, total protein, cholesterol, triglycerides and CO2. It's important to note that despite significant shifts occurring between the 2nd and 3rd trimester, there was no significant change in platelets, hematocrit, hemoglobin, lymphocytes or CO2 between non-pregnant and 3rd trimester blood samples. The normal reference ranges for each trimester established herein, will enable future identification of abnormalities occurring during pregnancy and help improve our understanding of factors potentially influencing a failed or successful pregnancy outcome.

Prevalence of Urogenital Carcinoma in Stranded California Sea Lions ( Zalophus californianus) from 2005-15.

Urogenital carcinoma is common in wild California sea lions ( Zalophus californianus) along the west coast of the US. From 1979 to 1994, this cancer was observed in 18% (66/370) of necropsied subadult and adult sea lions at The Marine Mammal Center in Sausalito, California. A retrospective review of records from 1 January 2005 to 31 December 2015 was performed to characterize prevalence and characteristics of cancer over this decade. Fourteen percent (263/1917) of necropsied sea lions had cancer, of which 90% (237/263) were urogenital carcinoma. The prevalence of urogenital carcinoma was significantly higher in adults compared to juveniles and subadults. Advanced-stage disease with metastases was identified histologically in 78% (182/232) of cases and was the cause of death in 95% (172/182) of these cases. Metastases were most common in lung and lymph nodes, and hydronephrosis, secondary to ureter obstruction by metastases, was identified in 62% (114/185) of animals with advanced disease. No significant temporal change in prevalence was detected over the decade, and this highly aggressive, fatal cancer remains common in stranded California sea lions.