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.

Ventilation and gas exchange before and after voluntary static surface breath-holds in clinically healthy bottlenose dolphins, Tursiops truncatus.

We measured respiratory flow (V̇), breathing frequency (fR), tidal volume (VT), breath duration and end-expired O2 content in bottlenose dolphins (Tursiops truncatus) before and after static surface breath-holds ranging from 34 to 292 s. There was considerable variation in the end-expired O2, VT and fR following a breath-hold. The analysis suggests that the dolphins attempt to minimize recovery following a dive by altering VT and fR to rapidly replenish the O2 stores. For the first breath following a surface breath-hold, the end-expired O2 decreased with dive duration, while VT and fR increased. Throughout the recovery period, end-expired O2 increased while the respiratory effort (VT, fR) decreased. We propose that the dolphins alter respiratory effort following a breath-hold according to the reduction in end-expired O2 levels, allowing almost complete recovery after 1.2 min.

Initial characterization of novel beaked whale morbillivirus in Hawaiian cetaceans.

Cetacean morbillivirus (CeMV) is a causative factor in epizootics that have resulted in thousands of deaths throughout the Atlantic and Mediterranean since 1987, but less is known of its presence and significance in the Pacific. The first case of CeMV reported in Hawai'i was in a Longman's beaked whale that stranded in 2010. The initial CeMV sequence from this individual indicated the possibility of a novel strain. To address this, archived samples from cetaceans that stranded in Hawai'i between 1997 and 2014 were screened for CeMV. The beaked whale morbillivirus (BWMV) was detected in 15 individuals representing 12 different species (24% of Code 1 and 2 stranded cetaceans). The earliest detected case was a humpback whale that stranded in 1998. Sequence comparisons of a 2.2 kb sequence spanning the phosphoprotein (P) and nucleocapsid (N) genes strongly suggest that the BWMV represents a novel strain of CeMV present in Hawai'i and the Central Pacific. In contrast to recently reported isolates from Brazil and Australia that may represent a distinct clade, BWMV appears to be more closely related to known strains of CeMV (dolphin morbillivirus; porpoise morbillivirus; and pilot whale morbillivirus). Detection rates with repeat sampling of positive lymph nodes were between 2 and 61%, illustrating the extreme heterogeneity that can occur in affected tissues. Taken together, these results suggest that BWMV may be common and established in Hawaiian cetacean populations. BWMV will be important for understanding CeMV and health threats in the relatively understudied cetaceans of the Pacific.

Lung mechanics and pulmonary function testing in cetaceans.

We measured esophageal pressures, respiratory flow rates, and expired O2 and CO2 in six adult bottlenose dolphins (Tursiops truncatus) during voluntary breaths and maximal (chuff) respiratory efforts. The data were used to estimate the dynamic specific lung compliance (sCL), the O2 consumption rate (V̇O2 ) and CO2 production rates (V̇CO2 ) during rest. Our results indicate that bottlenose dolphins have the capacity to generate respiratory flow rates that exceed 130 l s(-1) and 30 l s(-1) during expiration and inspiration, respectively. The esophageal pressures indicated that expiration is passive during voluntary breaths, but active during maximal efforts, whereas inspiration is active for all breaths. The average sCL of dolphins was 0.31±0.04 cmH2O(-1), which is considerably higher than that of humans (0.08 cmH2O(-1)) and that previously measured in a pilot whale (0.13 cmH2O(-1)). The average estimated V̇O2  and V̇CO2  using our breath-by-breath respirometry system ranged from 0.857 to 1.185 l O2 min(-1) and 0.589 to 0.851 l CO2 min(-1), respectively, which is similar to previously published metabolic measurements from the same animals using conventional flow-through respirometry. In addition, our custom-made system allows us to approximate end tidal gas composition. Our measurements provide novel data for respiratory physiology in cetaceans, which may be important for clinical medicine and conservation efforts.

Coinfection and vertical transmission of Brucella and Morbillivirus in a neonatal sperm whale (Physeter macrocephalus) in Hawaii, USA.

The viral genus Morbillivirus and the bacterial genus Brucella have emerged as important groups of pathogens that are known to affect cetacean health on a global scale, but neither pathogen has previously been reported from endangered sperm whales (Physeter macrocephalus). A female neonate sperm whale stranded alive and died near Laie on the island of Oahu, Hawaii, US, in May of 2011. Congestion of the cerebrum and enlarged lymph nodes were noted on the gross necropsy. Microscopic findings included lymphoid depletion, chronic meningitis, and pneumonia, suggesting an in utero infection. Cerebrum, lung, umbilicus, and select lymph nodes (tracheobronchial and mediastinal) were positive for Brucella by PCR. Brucella sp. was also cultured from the cerebrum and from mediastinal and tracheobronchial lymph nodes. Twelve different tissues were screened for Morbillivirus by reverse-transcriptase (RT)-PCR and select tissues by immunohistochemistry, but only the tracheobronchial lymph node and spleen were positive by RT-PCR. Pathologic findings observed were likely a result of Brucella, but Morbillivirus may have played a key role in immune suppression of the mother and calf. The in utero infection in this individual strongly supports vertical transmission of both pathogens.

Audiogram of a stranded Blainville's beaked whale (Mesoplodon densirostris) measured using auditory evoked potentials.

Quantifying and understanding the impact of anthropogenic sound on marine mammals has been the focus of many researchers both in laboratory settings as well as in the field. This study presents the audiogram of a sub-adult Blainville's beaked whale that stranded in Hawaii. The hearing measurements were conducted using the non-invasive auditory brainstem response technique. A total of 11 sinusoidally amplitude modulated tones were tested ranging from 5.6 to 160 kHz. The audiogram data indicated that the region of best hearing was found between 40 and 50 kHz with thresholds below 50 dB. This frequency range partially overlaps with the frequency modulated upsweep that Blainville's beaked whales have been reported to use during echolocation. These results match the frequency range obtained from the hearing measurements of a Gervais' beaked whale previously tested using contact acoustic stimulation and emphasize the importance of obtaining rapid hearing measurements on live stranded animals to improve the understanding of poorly known species.

Fetal echocardiographic evaluation of the bottlenose dolphin (Tursiops truncatus).

In humans, fetal echocardiography represents the most important tool for the assessment of the cardiovascular well-being of the fetus. However, because of logistic, anatomic, and behavioral challenges, detailed fetal echocardiographic evaluation of marine mammals has not been previously described. Because the application of fetal echocardiography to cetaceans could have both clinical and academic importance, an approach to evaluating the fetal dolphin's cardiovascular status was developed with conventional, fetal echocardiographic techniques developed in humans. Eight singleton fetal bottlenose dolphins (Tursiops truncatus) were evaluated, each between 6 and 11 mo gestation; six fetuses underwent two fetal echocardiographic evaluations each, four at 3-mo intervals, and two at 0.5-mo intervals. Evaluations were performed without sedation, using conventional, portable ultrasound systems. Multiple transducers, probes, and maternal dolphin positions were used to optimize image quality. Fetal echocardiography included two-dimensional imaging and color flow mapping of the heart and great arteries, as well as pulsed Doppler evaluation of the umbilical artery and vein. Thorough evaluations of the fetal dolphins' cardiovascular status were performed, with the greatest resolution between 8 and 9 mo gestation. With the use of published human fetal echocardiographic findings for comparison, fetal echocardiography demonstrated normal structure and function of the heart and great arteries, including the pulmonary veins, inferior vena cava, right and left atria, foramen ovale, tricuspid and mitral valves, right and left ventricles, ventricular septum, pulmonary and aortic valves, main pulmonary artery and ascending aorta, and ductus arteriosus. Pulsed Doppler techniques demonstrated normal umbilical arterial and venous waveforms, and color flow mapping demonstrated absence of significant valvar regurgitation. Fetal echocardiography, particularly between 8 and 9 mo gestation, can provide a safe and detailed assessment of the cardiovascular status of the fetal bottlenose dolphin.

Cryptococcus gattiivgi in a spinner dolphin (Stenella longirostris) from Hawaii.

A spinner dolphin (Stenella longirostris) was found stranded in Hawaii with cutaneous nodules and enlarged lymph nodes. Numerous Cryptococcus gattii VGI yeast were observed in multiple organs with minimal inflammation. This case represents the first reported infection of C. gattii in a dolphin from Hawaii.

Echocardiographic evaluation of the bottlenose dolphin (Tursiops truncatus).

Safe and effective echocardiography would represent a valuable tool for marine mammal veterinarians and physiologists evaluating the dolphin heart. Unfortunately, conventional ultrasound technology (transthoracic echocardiography) has been limited by logistic, anatomic, and behavioral challenges. Five mature male Atlantic bottlenose dolphins (Tursiops truncatus) were trained for echocardiographic imaging (four for both transthoracic and transesophageal imaging, and one for only transthoracic imaging). It was noted that transesophageal image quality transiently improved when the dolphins spontaneously exhaled. Subsequently, dolphins were conditioned to hold their breath following forced exhalation, and imaging proceeded during such behavioral breath holds. Over 25 transthoracic and 100 transesophageal echocardiographic studies were performed, including both two-dimensional imaging and color flow mapping. Transthoracic imaging yielded poor-quality images of only small portions of the heart. In contrast, transesophageal imaging, which improved dramatically with behavioral breath holding following exhalation, yielded consistently high-quality images of the entire heart (mitral, tricuspid, aortic, and pulmonary valves, atrial and ventricular septa, left and right atria, left and right ventricles, and ascending aorta and main pulmonary artery). Color flow mapping demonstrated mild tricuspid regurgitation in all dolphins, and mild aortic regurgitation in one dolphin found to have a pedunculated mass arising from the sinutubular junction just above the aortic valve. There were no complications in nonsedated dolphins. The heart of the bottlenose dolphin can be safely, effectively, and reproducibly evaluated with the use of transesophageal echocardiography in conjunction with behavioral breath holding following forced exhalation. This approach, and the normative echocardiographic data generated from this work, lays the foundation for future echocardiographic studies of cetaceans.

Congenital diffuse hyperplastic goiter associated with perinatal mortality in 11 captive-born bottlenose dolphins (Tursiops truncatus).

Diffuse hyperplastic goiter was diagnosed by histopathology in 11 perinatal bottlenose dolphins (Tursiops truncatus) that died at four separate zoos and aquaria. Thyroid morphology of these animals was compared with the histologically normal thyroids of two stranded wild bottlenose dolphin calves, a neonate and a 2-mo-old calf. Histologic changes included reduced follicular luminal diameter, markedly reduced or absent luminal colloid, hypertrophy of follicular epithelium, and follicular dysplasia. The etiology of the thyroid gland lesion was not identified. Cause of death was not determined for most of these animals, but they were presumed to have died from metabolic derangements associated with the thyroid lesion, drowning, or dystocia.