The genome sequence of the killer whale, Orcinus orca (Linnaeus, 1758).

We present a genome assembly from an individual female Orcinus orca (the killer whale; Chordata; Mammalia; Artiodactyla; Delphinidae). The genome sequence is 2.65 gigabases in span. The majority of the assembly (93.76%) is scaffolded into 22 chromosomal pseudomolecules with the X sex chromosome assembled. The complete mitochondrial genome was also assembled and is 16.4 kilobases in length.

Is Harbor Porpoise (Phocoena phocoena) Exhaled Breath Sampling Suitable for Hormonal Assessments?

Over the last decades, exhaled breath sampling has been established for laboratory analysis in various cetacean species. Due to their small size, the usability of respiratory vapor for hormone assessments was questionable in harbor porpoises (Phocoena phocoena). This pilot study compared three different blow collection devices for their suitability in the field and during laboratory processing: a sterile petri dish covered by a Nitex membrane, as well as sterile 50 mL centrifuge tubes with or without manganese(II) chloride as a stabilizer. Collected exhales varied between three, five or ten, depending on feasibility. Hormones were extracted through an ether mix, followed by centrifugal evaporation and cortisol analysis using an immunoassay. Although close to the lower end of the assay's dynamic range, the ELISA produced results (n = 110, 0.102-0.937 ng/mL). Hence, a simple 50 mL centrifuge tube was determined as the best suited blow collection device, while three consecutive exhales proved sufficient to yield results. These findings are promising regarding the suitability of exhaled breath as a matrix for future endocrine and immune system-related studies in harbor porpoises. If further advanced, blow sampling can become an important, non-invasive tool for studying and monitoring health, stress levels and diseases in harbor porpoises.

Lungworm infections in harbour porpoises (Phocoena phocoena) in the German Wadden Sea between 2006 and 2018, and serodiagnostic tests.

Pseudaliid lungworm (Metastrongyloidea) infections and associated secondary bacterial infections may severely affect the health status of harbour porpoises (Phocoena phocoena) in German waters. The presented retrospective analysis including data from 259 harbour porpoises stranded between 2006 and 2018 on the German federal state of Schleswig-Holstein's North Sea coast showed that 118 (46%) of these stranded individuals harboured a lungworm infection. During this 13-year period, a significant difference in annual lungworm prevalence was only observed between the years 2006 and 2016. Lungworm coinfections of bronchi and pulmonary blood vessels were observed in 85.6% of positive cases. Mild infection levels were detected in 22.9% of infected animals and were most common in the age class of immature individuals (74.1%). Moderate and severe infections were present in 38.1% and 39.0% of the lungworm positive animals, respectively. Their distribution in immatures (51.1% and 54.3%) and adults (48.9% and 43.4%) did not show significant differences. In stranded animals, lungworm diagnosis can be easily obtained via necropsy, while reliable lungworm diagnosis in living porpoises requires invasive bronchoscopy or faecal examination, which is difficult to obtain in cetaceans. To overcome this issue, an enzyme-linked immunosorbent assay (ELISA) and immunoblot based on recombinant major sperm protein (MSP) of the cattle lungworm were evaluated as potential diagnostic tools in harbour porpoises. However, in contrast to hitherto other investigated host species, no reliable antibody response pattern was detectable in harbour porpoise serum/plasma or whole blood samples. Thus, MSP-based serological tests are considered unsuitable for lungworm diagnosis in harbour porpoises.

Haematology and clinical blood chemistry in harbour porpoises (Phocoena phocoena) from the inner Danish waters.

Harbour porpoises (Phocoena phocoena) in the Baltic Sea are under increasing pressure from anthropogenic activities, which affect the overall health of populations. Individuals' haematologic and biochemistry parameters are known to be linked to a population's health status and are therefore useful tools for cross-population comparisons and to assess health patterns of a population through time. However, it is often difficult to acquire data representing the full range of values and assess the influence of intrinsic and extrinsic factors. Here, we present the range of haematology and blood chemistry values obtained from 46 wild (n = 54 blood samples) individuals incidentally caught in pound-nets and five porpoises in long-term human care (n = 449 blood samples) from an outdoor semi-open facility in Denmark. Although it was not possible to formally test the differences between samples from free-ranging and captive individuals, lymphocyte values were lower for free-ranging animals whereas eosinophils and white blood cell values were higher in captive individuals. Aspartate aminotransferase and alanin aminotransferase values were also lower for captive individuals compared to free-ranging ones. Age group did not influence any of the blood parameters tested for free-ranging individuals. Sodium values were higher for males compared to females. Values were higher and lower in the fall for platelets and lactic acid dehydrogenase, respectively, compared to the other seasons. Based on samples yielded by individuals in long-term human care, haemoglobin, mean cell volume, white blood cells, absolute lymphocyte count, and alkaline phosphatase values were all influenced by health status based on clinical examination. These are therefore candidate parameters to assess health status of wild porpoises. Our results underline that it is essential to obtain ranges of reference values for all haematologic and biochemistry markers in order to assess health status of free-ranging individuals. Individuals in human care provide the opportunity to observe biological and ecological determinates (e.g. age, season) of long-term biomarker response patterns and to assess the suite of biomarkers best suited to predict individual health status.

Clinical, pathological, and laboratory diagnoses of diseases of harbour porpoises (Phocoena phocoena), live stranded on the Dutch and adjacent coasts from 2003 to 2016.

Harbour porpoises (Phocoena phocoena) in the North Sea live in an environment heavily impacted by humans, the consequences of which are a concern for their health. Autopsies carried out on stranded harbour porpoises provide an opportunity to assess health problems in this species. We performed 61 autopsies on live-stranded harbour porpoises, which died following admission to a rehabilitation centre between 2003 and 2016. The animals had stranded on the Dutch (n = 52) and adjacent coasts of Belgium (n = 2) and Germany (n = 7). We assigned probable causes for stranding based on clinical and pathological criteria. Cause of stranding was associated in the majority of cases with pathologies in multiple organs (n = 29) compared to animals with pathologies in a single organ (n = 18). Our results show that the three most probable causes of stranding were pneumonia (n = 35), separation of calves from their mother (n = 10), and aspergillosis (n = 9). Pneumonia as a consequence of pulmonary nematode infection occurred in 19 animals. Pneumonia was significantly associated with infection with Pseudalius inflexus, Halocercus sp., and Torynurus convolutus but not with Stenurus minor infection. Half of the bacterial pneumonias (6/12) could not be associated with nematode infection. Conclusions from this study are that aspergillosis is an important probable cause for stranding, while parasitic infection is not a necessary prerequisite for bacterial pneumonia, and approximately half of the animals (29/61) probably stranded due to multiple causes. An important implication of the observed high prevalence of aspergillosis is that these harbour porpoises suffered from reduced immunocompetence.

Dolphins' Willingness to Participate (WtP) in Positive Reinforcement Training as a Potential Welfare Indicator, Where WtP Predicts Early Changes in Health Status.

Welfare science has built its foundations on veterinary medicine and thus measures of health. Since bottlenose dolphins (Tursiops truncatus) tend to mask symptoms of poor health, management in captivity would benefit from advanced understanding on the links between health and behavioural parameters, and few studies exist on the topic. In this study, four representative behavioural and health measures were chosen: health status (as qualified by veterinarians), percentage of daily food eaten, occurrences of new rake marks (proxy measure of social activity), and willingness to participate (WtP) in Positive Reinforcement Training sessions as qualitatively measured by their caretakers. These data were collected multiple times a day, every day over the course of a year from a multi-facility, large sample size (n dolphins = 51), allowing powerful analyses of the relationships between measures. First, it was found that dolphins with a higher WtP score also had a significantly better health status, ate a higher percentage of their daily food, and a lower occurrence of new rake marks. In addition, the WtP score was significantly lower up to 3 days before the weekly veterinary diagnosis of a decrease in health state; the percentage of daily food eaten and new rake mark measures did not show any significant change before such a diagnosis. These results suggest that WtP in training sessions is a potential behavioural measure of dolphin welfare, and an indicator of early changes in the dolphins' health state. We therefore suggest measurement of WtP as a more practical and non-invasive tool to support veterinary care and general management. More work needs to be conducted to elucidate the influence of social behaviour on health, and to identify other potential welfare indicators, but this long-term study has shown that qualitative measures can be both practical and valid when assessing dolphin welfare.

Successful treatment of azole-resistant invasive aspergillosis in a bottlenose dolphin with high-dose posaconazole.

Invasive aspergillosis due to azole-resistant Aspergillus fumigatus is difficult to manage. We describe a case of azole-resistant invasive aspergillosis in a female bottlenose dolphin, who failed to respond to voriconazole and posaconazole therapy. As intravenous therapy was precluded, high dose posaconazole was initiated aimed at achieving trough levels exceeding 3 mg/l. Posaconazole serum levels of 3-9.5 mg/l were achieved without significant side-effects. Follow-up bronchoscopy and computed tomography showed complete resolution of the lesions.

Echocardiographic diagnosis and necropsy findings of a congenital ventricular septal defect in a stranded harbor porpoise.

A live-stranded harbor porpoise Phocoena phocoena was found on the west coast of the Dutch island Texel (North Sea) and transported to a rehabilitation center for small cetaceans, where it underwent a veterinary health check. Cardiac auscultation revealed a systolic cardiac murmur with the point of maximal intensity in the right hemithorax with an intensity of IV out of VI. Transthoracic echocardiography revealed a congenital ventricular septal defect with left-to-right shunting. Because the left atrium was not dilated according to the reference range of canine left atrium to aortic ratio, the presence of congestive heart failure was considered very unlikely. Therefore, this congenital cardiac anomaly was thought to be a clinically non-relevant incidental finding and would not explain the weakness, coughing, anorexia, vomiting, and diarrhea. Because the animal was still unable to swim or eat by itself after 2 wk of supportive care, it was euthanized. Post-mortem examination confirmed the presence of a ventricular septal defect. The weight of the heart relative to the animal's length was greater than expected, using linear regression analysis on the lengths and cardiac weights of 71 other stranded wild harbor porpoises without macroscopic cardiac pathologic changes. This finding suggests that the left ventricle had an eccentric hypertrophy because of volume overload resulting from the intracardiac shunt. This is the first report of a congenital cardiac anomaly and its ante-mortem diagnosis in this species. Data presented for the other 71 harbor porpoises may provide reference values for this species.

No serological evidence that harbour porpoises are additional hosts of influenza B viruses.

Influenza A and B viruses circulate among humans causing epidemics almost annually. While various hosts for influenza A viruses exist, influenza B viruses have been detected only in humans and seals. However, recurrent infections of seals in Dutch coastal waters with influenza B viruses that are antigenetically distinct from influenza B viruses circulating among humans suggest that influenza B viruses have been introduced into this seal population by another, non-human, host. Harbour porpoises (Phocoena phocoena) are sympatric with seals in these waters and are also occasionally in close contact with humans after stranding and subsequent rehabilitation. In addition, virus attachment studies demonstrated that influenza B viruses can bind to cells of the respiratory tract of these animals. Therefore, we hypothesized that harbour porpoises might be a reservoir of influenza B viruses. In the present study, an unique set of serum samples from 79 harbour porpoises, stranded alive on the Dutch coast between 2003 and 2013, was tested for the presence of antibodies against influenza B viruses by use of the hemagglutination inhibition test and for antibodies against influenza A viruses by use of a competitive influenza A nucleoprotein ELISA. No antibodies were detected against either virus, suggesting that influenza A and B virus infections of harbour porpoises in Dutch coastal waters are not common, which was supported by statistical analysis of the dataset.

Audiogram of a harbor porpoise (Phocoena phocoena) measured with narrow-band frequency-modulated signals.

The underwater hearing sensitivity of a two-year-old harbor porpoise was measured in a pool using standard psycho-acoustic techniques. The go/no-go response paradigm and up-down staircase psychometric method were used. Auditory sensitivity was measured by using narrow-band frequency-modulated signals having center frequencies between 250 Hz and 180 kHz. The resulting audiogram was U-shaped with the range of best hearing (defined as 10 dB within maximum sensitivity) from 16 to 140 kHz, with a reduced sensitivity around 64 kHz. Maximum sensitivity (about 33 dB re 1 microPa) occurred between 100 and 140 kHz. This maximum sensitivity range corresponds with the peak frequency of echolocation pulses produced by harbor porpoises (120-130 kHz). Sensitivity falls about 10 dB per octave below 16 kHz and falls off sharply above 140 kHz (260 dB per octave). Compared to a previous audiogram of this species (Andersen, 1970), the present audiogram shows less sensitive hearing between 2 and 8 kHz and more sensitive hearing between 16 and 180 kHz. This harbor porpoise has the highest upper-frequency limit of all odontocetes investigated. The time it took for the porpoise to move its head 22 cm after the signal onset (movement time) was also measured. It increased from about 1 s at 10 dB above threshold, to about 1.5 s at threshold.