AUDITORY EVOKED POTENTIALS AND BEHAVIORAL CONSIDERATIONS WITH HEARING LOSS IN SMALL CETACEANS: APPLICATION AS A STANDARD DIAGNOSTIC TEST IN HEALTH ASSESSMENT.

The primary sense in odontocetes is hearing and a large portion of the odontocete brain is devoted to the auditory processing of echolocation signals. Hearing deficits in odontocetes potentially compromise the ability to forage, navigate, socialize, and evade predators. This presents a challenge to survival and reproduction in wild odontocetes and can affect the general welfare of odontocetes under human care. Currently, little empirical information on how odontocete behavior is affected by hearing loss exists. This study investigated hearing deficits in several species of stranded dolphins and age-related hearing deficits in dolphins kept under human care through auditory evoked potential (AEP) testing and evaluated whether individual behavior correlated with hearing impairment. Behavioral questionnaires for participating animals were completed by individuals with extensive knowledge of the animals' history and behavior. A chi-square analysis determined whether animals with hearing impairment demonstrated behaviors that differed significantly from those considered normal. All tested individuals under human care over 35 years of age had some degree of hearing loss, as did a large percentage of previously stranded animals. Individuals with hearing loss exhibited a range of behavioral changes, including delays in learning new behaviors, accepting novel enrichment, and habituating to new environments. Some individuals with profound hearing loss also displayed a change in vocalization rate in various situations. Findings within previously stranded animals suggest AEP studies should be conducted in all stranded individuals entering rehabilitation. It is further recommended that dolphins living under human care undergo hearing tests as part of their normal health assessments, with emphasis on aging individuals and animals that exhibit delayed learning, respond poorly to audible cues, or show atypical vocalization behavior.

Survey of antibiotic-resistant bacteria isolated from bottlenose dolphins Tursiops truncatus in the southeastern USA.

Contamination of coastal waters can carry pathogens and contaminants that cause diseases in humans and wildlife, and these pathogens can be transported by water to areas where they are not indigenous. Marine mammals may be indicators of potential health effects from such pathogens and toxins. Here we isolated bacterial species of relevance to humans from wild bottlenose dolphins Tursiops truncatus and assayed isolated bacteria for antibiotic resistance. Samples were collected during capture-release dolphin health assessments at multiple coastal and estuarine sites along the US mid-Atlantic coast and the Gulf of Mexico. These samples were transported on ice and evaluated using commercial systems and aerobic culture techniques routinely employed in clinical laboratories. The most common bacteria identified were species belonging to the genus Vibrio, although Escherichia coli, Shewanella putrefaciens, and Pseudomonas fluorescens/putida were also common. Some of the bacterial species identified have been associated with human illness, including a strain of methicillin-resistant Staphylococcus aureus (MRSA) identified in 1 sample. Widespread antibiotic resistance was observed among all sites, although the percentage of resistant isolates varied across sites and across time. These data provide a baseline for future comparisons of the bacteria that colonize bottlenose dolphins in the southeastern USA.

Behavior Skills Training with Zoological Staff to Increase Killer Whale Attending Behavior.

Attending is a requisite behavior for interactions between animals in zoological care and their care specialist (trainers). Head-dropping behavior (HDB) is incompatible with attending and was observed with two killer whales during whale-trainer interactions (WTI). Initial observations also noted inconsistencies in the trainers' application of shaping procedures during WTI. A three-part plan was developed to address trainers procedural fidelity, increase whale attending during WTI, and program for the behavioral generalization and maintenance of the whale-trainer dyads (dyad). First, behavior skills training (BST) was used to instruct trainers on discrete trial training (DTT). Second, the trainers' applied their acquired skill of DTT, targeting attending behavior, with their whale during WTI. Third, behavioral generalization was programed by switching the dyad pairs in an additional DTT generalization phase. The findings demonstrated a strong positive correlation between trainer DTT fidelity and whale attending. Finally, the results suggest that future assessments of behavioral management programs with zoological trainers and animals are warranted.

A novel case of Fusarium oxysporum infection in an Atlantic bottlenose dolphin (Tursiops truncatus).

A necropsy was performed on a captive-born, 10-yr-old male Atlantic bottlenose dolphin (Tursiops truncatus) after it died acutely. Gross necropsy findings revealed hemorrhage within the right cerebrum, right cerebellum, and right eye. Histopathologic findings revealed a moderate multifocal acute necrotizing meningoencephalitis with intralesional fungal hyphae. Several pieces of cerebrum and cerebellum and cerebrospinal fluid were sent to the Fungus Testing Laboratory in San Antonio, Texas (U.S.A.). The culture yielded Fusarium oxysporum, which was confirmed by internal transcribed spacer and D1-D2 sequencing. Fusarium oxysporum infection has been reported in marine mammals. No cases of noncutaneous F. oxysporum infection in a cetacean that was not on long-term antimicrobials have been reported in the literature.

Caryospora-Like Coccidia Infecting Green Turtles (Chelonia mydas): An Emerging Disease With Evidence of Interoceanic Dissemination.

Protozoa morphologically consistent with Caryospora sp. are one of the few pathogens associated with episodic mass mortality events involving free-ranging sea turtles. Parasitism of green turtles (Chelonia mydas) by these coccidia and associated mortality was first reported in maricultured turtles in the Caribbean during the 1970s. Years later, epizootics affecting wild green turtles in Australia occurred in 1991 and 2014. The first clinical cases of Caryospora-like infections reported elsewhere in free-ranging turtles were from the southeastern US in 2012. Following these initial individual cases in this region, we documented an epizootic and mass mortality of green turtles along the Atlantic coast of southern Florida from November 2014 through April 2015 and continued to detect additional, sporadic cases in the southeastern US in subsequent years. No cases of coccidial disease were recorded in the southeastern US prior to 2012 despite clinical evaluation and necropsy of stranded sea turtles in this region since the 1980s, suggesting that the frequency of clinical coccidiosis has increased here. Moreover, we also recorded the first stranding associated with infection by a Caryospora-like organism in Hawai'i in 2018. To further characterize the coccidia, we sequenced part of the 18S ribosomal and mitochondrial cytochrome oxidase I genes of coccidia collected from 62 green turtles found in the southeastern US and from one green turtle found in Hawai'i. We also sequenced the ribosomal internal transcribed spacer regions from selected cases and compared all results with those obtained from Caryospora-like coccidia collected from green turtles found in Australia. Eight distinct genotypes were represented in green turtles from the southeastern US. One genotype predominated and was identical to that of coccidia collected from the green turtle found in Hawai'i. We also found a coccidian genotype in green turtles from Florida and Australia with identical 18S and mitochondrial sequences, and only slight inter-regional differences in the internal transcribed spacer 2. We found no evidence of geographical structuring based on phylogenetic analysis. Low genetic variability among the coccidia found in green turtle populations with minimal natural connectivity suggests recent interoceanic dissemination of these parasites, which could pose a risk to sea turtle populations.

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.

Demographic clusters identified within the northern Gulf of Mexico common bottlenose dolphin (Tursiops truncates) unusual mortality event: January 2010-June 2013.

A multi-year unusual mortality event (UME) involving primarily common bottlenose dolphins (Tursiops truncates) was declared in the northern Gulf of Mexico (GoM) with an initial start date of February 2010 and remains ongoing as of August 2014. To examine potential changing characteristics of the UME over time, we compared the number and demographics of dolphin strandings from January 2010 through June 2013 across the entire GoM as well as against baseline (1990-2009) GoM stranding patterns. Years 2010 and 2011 had the highest annual number of stranded dolphins since Louisiana's record began, and 2011 was one of the years with the highest strandings for both Mississippi and Alabama. Statewide, annual numbers of stranded dolphins were not elevated for GoM coasts of Florida or Texas during the UME period. Demographic, spatial, and temporal clusters identified within this UME included increased strandings in northern coastal Louisiana and Mississippi (March-May 2010); Barataria Bay, Louisiana (August 2010-December 2011); Mississippi and Alabama (2011, including a high prevalence and number of stranded perinates); and multiple GoM states during early 2013. While the causes of the GoM UME have not been determined, the location and magnitude of dolphin strandings during and the year following the 2010 Deepwater Horizon oil spill, including the Barataria Bay cluster from August 2010 to December 2011, overlap in time and space with locations that received heavy and prolonged oiling. There are, however, multiple known causes of previous GoM dolphin UMEs, including brevetoxicosis and dolphin morbillivirus. Additionally, increased dolphin strandings occurred in northern Louisiana and Mississippi before the Deepwater Horizon oil spill. Identification of spatial, temporal, and demographic clusters within the UME suggest that this mortality event may involve different contributing factors varying by location, time, and bottlenose dolphin populations that will be better discerned by incorporating diagnostic information, including histopathology.

Absence of humoral response in flamingos and red-tailed hawks to experimental vaccination with a killed West Nile virus vaccine.

Sixteen Chilean flamingos, Phoenicopterus chiles, and 10 red-tailed hawks, Buteo jamacensis, were vaccinated in the pectoral muscle with 0.2 ml of a commercially produced killed West Nile virus vaccine intended for use in horses. Half the birds of each species received a booster vaccination 3 weeks after the first injection. Three weeks after the booster vaccination, none of 13 birds surveyed had detectable antibody to West Nile virus.

Hypocitraturia in common bottlenose dolphins (Tursiops truncatus): assessing a potential risk factor for urate nephrolithiasis.

Numerous cases of urate nephrolithiasis in managed collections of common bottlenose dolphins (Tursiops truncatus) have been reported, but nephrolithiasis is believed to be uncommon in wild dolphins. Risk factors for urate nephrolithiasis in humans include low urinary pH and hypocitraturia. Urine samples from 94 dolphins were collected during April 2006 through June 2009 from 4 wild populations (n = 62) and 4 managed collections (n = 32). In addition, urine uric acid and pH were tested in a subset of these animals. Our null hypothesis was that wild and managed collection dolphins would have no significant differences in urinary creatinine, citrate, and uric acid concentrations and pH. Among urine samples from all 94 dolphins, the urinary levels (mean +/- SEM) for creatinine, citrate, uric acid, and pH were 139 +/- 7.6 mg/dL, 100 +/- 20 mg citrate/g creatinine, 305 +/- 32 mg uric acid/g creatinine, and 6.2 +/- 0.05, respectively. Of the 4 urinary variables, only citrate concentration varied significantly between the 2 primary study groups; compared with wild dolphins, managed collection dolphins were more likely to have undetectable levels of citrate in the urine (21.0% and 81.3%, respectively). Mean urinary citrate concentrations for managed collection and wild dolphin populations were 2 and 150 mg citrate/g creatinine, respectively. We conclude that some managed collections of dolphins, like humans, may be predisposed to urate nephrolithiasis due to the presence of hypocitraturia. Subsequent investigations can include associations between metabolic syndrome, hypocitraturia, and urate nephrolithiasis in humans and dolphins; and the impact of varying levels of seawater ingestion on citrate excretion.

Serological diagnosis of Brucella infections in odontocetes.

Brucella ceti causes disease in Odontoceti. The absence of control serum collections and the diversity of cetaceans have hampered the standardization of serological tests for the diagnosis of cetacean brucellosis. Without a "gold" standard for sensitivity and specificity determination, an alternative approach was followed. We designed an indirect enzyme-linked immunosorbent assay (iELISA) that recognizes immunoglobulins G (IgGs) from 17 odontocete species as a single group. For the standardization, we used Brucella melitensis and Brucella abortus lipopolysaccharides, serum samples from seven resident odontocetes with no history of infectious disease displaying negative rose bengal test (RBT) reactions, and serum samples from seven dolphins infected with B. ceti. We compared the performance of the iELISA with those of the protein G ELISA (gELISA), the competitive ELISA (cELISA), and the immunofluorescence (IF) and dot blot (DB) tests, using 179 odontocete serum samples and RBT as the reference. The diagnostic potential based on sensitivity and specificity of the iELISA was superior to that of gELISA and cELISA. The correlation and agreement between the iELISA and the gELISA were relatively good (R(i/g)2 = 0.65 and kappa(i/g) = 0.66, respectively), while the correlation and agreement of these two ELISAs with cELISA were low (R(i/c)2 = 0.46, R(g/c)2 = 0.37 and kappa(i/c) = 0.62, kappa(g/c) = 0.42). In spite of using the same anti-odontocete IgG antibody, the iELISA was more specific than were the IF and DB tests. An association between high antibody titers and the presence of neurological symptoms in dolphins was observed. The prediction is that iELISA based on broadly cross-reacting anti-dolphin IgG antibody would be a reliable test for the diagnosis of brucellosis in odontocetes, including families not covered in this study.