Nahiid Stephens

The rationale for clinical trials in veterinary research is clear. However, the rationale for cetacean health research may not be as obvious. Firstly, it adds important information to population biology, ecology and behavioural studies. Secondly, it informs conservation efforts, in particular work surrounding laws and guidelines. Thirdly, it allows scientists to connect to the public via education and outreach tools; such citizen science programs are both a means and an end, serving to engage the public, but also generating great baseline data that researchers can draw on. And, finally, cetaceans are marine and estuarine environmental sentinels. As long-lived apex predators, they accumulate organic and inorganic contaminants, particularly because they range within coastal and estuarine areas near human population centres. Cetaceans are therefore ‘biological sentinels,’ i.e. good indicators of mid- to long-term changes in their ecosystems and the overall health of that area. Thus, useful information regarding ecosystem health can be gained in studying cetacean health.

Cetacean Morbillivirus (CMV) has caused several epizootics in cetaceans globally. An unusual mortality event among Tursiops aduncus in Perth, Western Australia (WA) coincided with increased strandings statewide in 2009. Necropsy of two dolphins identified mycotic encephalitis and pneumonia as the cause of death, respectively. Morbilliviral antigen was detected in multiple tissues using immunohistochemistry and confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) using primers for morbilliviral N and P genes. Sequencing of amplified RT-PCR products (approximately 238 bp in length from the N gene & 425 bp from the P gene) confirmed the presence of CMV in both dolphins and facilitated phylogenetic analysis. The N and P genes from the WA dolphins had respectively, 79% - 83% and 75% - 79% nucleotide identity to the highly conserved N and P genes of various strains of CMV. There was only 79% - 83% identity between the N and P nucleotide sequences from the WA dolphins and a variant from a Tursiops truncatus that stranded in Queensland (QLD), Australia in 2010. This is the first report of CMV-related mortality for T. aduncus in Australian waters and for the Indian Ocean. Preliminary phylogenetic data suggest that the WA variant is distinct from all other morbilliviruses, and is closely related to, but divergent from, other cetacean morbilliviruses. It is distinct from the QLD CMV variant, which is more closely related to the CMV isolates from the Northern Hemisphere. Furthermore, the WA variant appears to be the most closely related marine mammal morbillivirus to the terrestrial genus members identified to date. Evidence of a divergent CMV has implications for understanding the evolutionary history of CMV and supports ongoing surveillance and archival sampling to elucidate the role of CMV and other pathogens in future mortalities.

Currently, there is no standardised method for classifying skin lesions on small cetaceans from image data taken during boat-based surveys. As there is no way to be certain of aetiology from an image alone, these systems must be primarily based on descriptive gross morphology. Many classification systems used in the literature are subjective and only encompass lesions that have been seen in their population. Hence, no studies use the same system making it difficult to compare lesion occurrence among populations. This study presents a standardised classification system for use on boat-based photographic survey data. A comprehensive literature review was undertaken to develop the classification system encompassing the previous classification of skin lesions in dolphins; known aetiologies; and morphology of the skin during infection, injury and healing. Images from bottlenose populations in the Swan-Canning Estuary, Mandurah and Bunbury were also examined. The classification system developed separates lesions into two main types: possibly infectious and obvious injuries. Possibly infectious lesion categories include Hyperpigmented, Hypopigmented, Targetoid, Raised and/or Proliferative, Depressed/Sunken, Concentric Rings, Tattoolike and Discolouration. Injury categories include Bite Wounds, Abrasions/Excoriations and Anthropogenic. This system reduces the subjectivity and unnecessary splitting of categories that occurs when using existing classification schemes and will make studies that employ it more comparable. This is important as lesion occurrence can reflect population and ecosystem health. The system has been tested on the Swan-Canning Estuary population using 4547 images taken during boat-based surveys from 26/7/2011 to 30/6/2013.

Purpose: The somatic neuromuscular junction (NMJ) is a valuable experimental model of synapse formation. Such experiments are most commonly performed on placental mammals (e.g. mouse), where all four limbs develop on a similar timescale. By contrast, some Australian marsupials have very segmented limb development, where forelimbs are precociously developed to assist the journey to the pouch, while hindlimbs are comparatively unformed. We have compared the morphological development of the NMJ in kangaroo hindlimbs and forelimbs, to investigate how NMJ development proceeds in an animal with this novel developmental pattern. Methods: Three Western Grey Kangaroo neonates aged postnatal day (P) 0, 26 and 100 were ethically sourced from licensed shooters in South-Western Australia. 5 forelimb and 4 hindlimb muscles from each specimen were embedded, cryosectioned, and stained immunohistochemically for NMJ compartment proteins. Results: Confocal micrographs of adult kangaroo NMJs revealed an unusual oval endplate with a internal perforation at its centre. When markers of NMJ development described in rodents (e.g. neurofilament distribution, ACh receptor clustering) were analysed in P0 neonates, forelimb NMJs were considerably more developed than hindlimb junctions. However, between P0 and 100, NMJs in kangaroo forelimbs developed at a slower rate than hindlimb NMJs, so that all limbs were similarly developed by P100. Conclusions: Our preliminary data indicates that the segmented limb development in Western Grey Kangaroos is reflected at the level of the NMJ. This segmented NMJ development, combined with the relative accessibility of kangaroo neonates (which undergo a large degree of maturation in the pouch), present novel opportunities for studying synaptic development compared with placental mammals.

The principles of synapse formation have been largely unraveled by investigations at the somatic neuromuscular junction (NMJ). Such experiments are commonly performed on placental mammals (e.g. mouse), where all four limbs develop on a similar timescale. By contrast, some Australian marsupials have very segmented limb development, where forelimbs are precociously developed to assist the journey to the pouch, while hindlimbs are comparatively unformed. We explored the implications of this novel developmental pattern for the morphological development of marsupial NMJs. Western Grey Kangaroo neonates (n = 8) aged postnatal day (P) 0-100 were ethically sourced from licensed shooters. Multiple (5-9) muscles from each specimen were embedded, cryosectioned, and stained immunohistochemically for NMJ compartment proteins. Confocal micrographs of adult kangaroo NMJs revealed small oval endplates with a central internal perforation. Standard indices of synapse maturation (e.g. ACh receptor clustering) confirmed significant NMJ development from P0-P100. Notably, forelimb NMJs were considerably more developed than hindlimb junctions at P0. From P0-100, kangaroo forelimb NMJs developed at a slower rate than hindlimb NMJs, so that all limbs were similarly developed by P100. Interestingly, within the hindlimb, a detailed comparison of 5 muscles suggested a single, homogenous developmental profile, in contrast with recent studies in mouse suggesting both fast and delayed synapsing muscle populations (Pun et al., 2002). The segmented nature of NMJ development in Western Grey Kangaroos combined with the relative accessibility provided by a protracted maturation in the pouch, suggest that marsupials may present novel opportunities for studying synapse maturation compared with placental mammals.

From June to October 2009 six bottlenose dolphins (Tursiops aduncus) were found dead in the Swan-Canning Estuary, Perth. Opportunistic bacterial and mycotic infections, severe ulcerative dermatitis, and lymphoid depletion suggested reduced immune competence. Ulcerative dermatitis was associated with intracytoplasmic eosinophilic inclusions typical of cetacean poxvirus. Morbillivirus antigen was detected by immunohistochemistry in lymphocytes, endothelium and biliary epithelium from two dolphins. We hypothesize that morbillivirus-induced immunosuppression exacerbated poxvirus dermatitis causing unusually severe ulcerative dermatitis and predisposed to bacterial and Aspergillus spp. infections. Blubber toxicology also found high levels of pollutants such as dieldrin (mean: 17.13 µg/g lipid). Temporal analysis of this event and historical records found an association between mortalities and seasonal flushing of the estuary by heavy spring rains and marked salinity declines. It appears that the Swan-Canning Estuary dolphin population is regulated by a balance between pathogens, environmental stressors and possibly, anthropogenic pollutants.

In 2009 six bottlenose dolphins (Tursiops aduncus) were found dead in the Swan Canning river system. Post-mortem findings suggested that the dolphins had impaired immune systems and further testing identified the presence of morbillivirus, tattoo skin disease and high contaminant burdens. Although morbillivirus is one of the most pathogenic viruses in cetaceans and has been implicated in marine mammal mortality events around the world, this is the first time the pathogen has been detected in marine mammals in Western Australia. How did the virus get here and what is its significance for dolphins in the Swan Canning river system? We will examine several theories regarding the presence and significance of morbillivirus infection. Regardless of the mechanism of introduction, it is rarely the case that wild animals are exposed to a single disease-causing agent at a time. Rather, the presentation of disease usually involves an interactive relationship between multiple disease-causing agents, the animal, and environmental factors. Thus, the 2009 dolphin mortalities are best understood as the result of a complex interplay of multiple factors with several significant knowledge gaps remaining.

In 2009 two adult female bottlenose dolphins were found dead within the Swan-Canning Estuary. Both dolphins had severe ulcerative skin lesions covering over 70% of their skin surface area. Histological analyses identified viral inclusion bodies characteristic of poxvirus within these skin lesions. The severity and extent of the lesions were deemed to have caused terminal debilitation in the two dolphins. Infection with dolphin poxvirus is usually characterised by one to several mild skin lesions in juveniles that regress with time. Poxvirus infection has never been documented as the cause of death in adult dolphins; furthermore the severe skin lesions observed in the two dolphins mark a novel presentation of dolphin poxvirus infection. The definitive mechanisms by which the characteristic poxvirus lesions may have progressed to the unusually severe lesions is currently unknown but possible factors may include: the presence of a poxvirus strain possessing greater virulence; and/or the potentially immunosuppressive effects associated with living in a heavily utilised and inherently stressful estuarine environment. Dolphins are recognised as biosentinels for coastal and estuarine ecosystems, and atypical disease presentations such as this may be indicative of the environmental conditions of systems like the Swan-Canning Estuary.

In 2009, an unprecedented number of humpback whales (Megaptera novaenangliae) (n=46) were found dead or dying on WA beaches. This compares to an average of less than five humpback whales each year in the preceding two decades. Speculation on causes of increased marine mammal mortalities usually focuses on starvation, disease or anthropogenic impacts, or some synergistic combination of these. The current situation is that very little is known about the health of the WA humpbacks in terms of their nutritional status, disease or level of adverse human interactions. We propose to aid in the investigation into increased mortalities by conducting opportunistic post-mortem examinations over a five year period. The project aims to: • identify and characterise factors associated with humpback whale strandings and; • determine baseline and epidemiological information on disease levels and the nutritional status of stranded humpback whales. The data and archived tissues collected will provide an invaluable baseline, thus allowing for the early detection of adverse impacts from natural and anthropogenic pressures and retrospective assessment for the incidence of currently unidentified or poorly-described pathogens.