Mark Flint

Postmortem Diagnostic Investigation of Disease in Free-Ranging Marine Turtle Populations: A Review of Common Pathologic Findings and Protocols

Over the past few decades, there have been increasing numbers of reports of diseases in marine turtles. Furthermore, in recent years, there have been documented instances of apparently new diseases emerging in these species of which the etiology and/or pathogenesis remain unknown. These instances i) raise concern for the survival of marine turtles, and ii) question the health and stability of the benthic marine environments in which turtles live. Knowledge of common disease processes and pathologic changes in lesions, along with a standardized approach to postmortem and sample collection are required to document and understand the host-agent-environment interactions in marine turtle health. This review combines, for the first time, a standardized approach to the postmortem of marine turtles for veterinary clinicians, with a concurrent descriptive review of the gross and microscopic pathologic changes in lesions commonly seen.

Reference intervals for plasma biochemical and hematologic measures in loggerhead sea turtles (Caretta Caretta) from Moreton bay Australia

Biochemical and hematologic reference intervals have been reported for loggerhead sea turtles (Caretta caretta, Linnaeus 1758), but low sample numbers and simple statistical analyses have constrained their diagnostic usefulness. During June 2007–May 2008, 101 loggerhead sea turtles in Moreton Bay, Queensland, Australia, were captured by hand from boats; clinically assessed to determine health status; blood was sampled; and biochemical and hematologic variables were measured. Of these turtles, 66 were classified as clinically healthy and 23 as unhealthy. Reference intervals were calculated using data from clinically healthy turtles. Of the clinically unhealthy turtles, 82 and 45% had at least one biochemical and hematologic result, respectively, outside of at least one of the calculated intervals. However, only low proportions of unhealthy loggerhead sea turtles had abnormal results for each variable. The highest percentage of unhealthy turtles that were outside at least one estimated reference interval was 35%, for thrombocyte counts. Neither sex nor maturity category (mature versus large immature) influenced the risk of being clinically unhealthy. These are the first plasma biochemical and hematologic reference intervals reported for loggerhead sea turtles from the southwestern Pacific Ocean. We conclude that, for loggerhead sea turtles in Moreton Bay, separate reference intervals are required for mature and immature turtles for thrombocyte counts and for male and female turtles for lymphocyte, heterophil, and total white cell counts; otherwise, a single reference interval can be used regardless of age or sex. When estimating reference intervals in loggerhead sea turtles, it is desirable to use both methods for calculating reference intervals used in this study because intervals can differ substantially between methods for some variables. Joint interpretation using reference intervals from both methods allows the categorization of results as “normal,” “suspect,” or “abnormal.”

Corneal Fibropapillomatosis in Green Sea Turtles (Chelonia mydas) in Australia.

Chelonid corneal fibropapillomatosis has not previously been recorded in Australian waters. During 2008, 724 green sea turtles (Chelonia mydas) were examined in Queensland, Australia at two sites, Moreton Bay (n=155) and Shoalwater Bay (n=569), during annual monitoring. In the same calendar year, 63 turtles were submitted from various sites in southern Queensland for post-mortem examination at the University of Queensland. Four of the 787 animals (0.5%) were found to have corneal fibropapillomas of varying size, with similar gross and microscopical features to those reported in other parts of the world. Two animals with corneal fibropapillomas also had cutaneous fibropapillomas. Clinical assessment indicated that these lesions had detrimental effects on the vision of the turtles and therefore their potential ability to source food, avoid predators and interact with conspecifics. Importantly, these findings represent an emergence of this manifestation of fibropapillomatosis in green sea turtle populations in the southern Pacific Ocean.

Clinical and Pathological Findings in Green Turtles ( Chelonia mydas ) from Gladstone, Queensland: Investigations of a Stranding Epidemic

An investigation into the health of green turtles was undertaken near Gladstone, Queensland, in response to a dramatic increase in stranding numbers in the first half of 2011. A total of 56 live turtles were subject to clinical examination and blood sampling for routine blood profiles, and 12 deceased turtles underwent a thorough necropsy examination. This population of green turtles was found to be in poor body condition and a range of infectious and non-infectious conditions were identified in the unhealthy turtles, including hepato-renal insufficiency (up to 81%, 27/33 based on clinical pathology), cachexia (92%, 11/12), parasitism (75%, 9/12), cardiopulmonary anomalies (42%, 5/12), gastroenteritis (25%, 3/12), masses (25%, 3/12) and mechanical impediments (17%, 2/12 based on necropsy). Overall, there was no evidence to indicate a unifying disease as a primary cause of the mass mortality. Recent adverse weather events, historic regional contamination and nearby industrial activities are discussed as potential causative factors.

BACTERIA ISOLATED FROM DUGONGS (DUGONG DUGON) SUB-MITTED FOR POSTMORTEM EXAMINATION IN QUEENSLAND, AUSTRALIA, 2000–2011

Abstract:  Microbial infection may contribute to disease in a significant proportion of marine mammal mortalities, but little is known about infectious bacterial species and their prevalence in dugongs (Dugong dugon). This study represents a survey of the species of bacteria and fungi isolated from dugongs submitted to the University of Queenslands School of Veterinary Science for postmortem examination. Thirty-six dugongs were included in the survey, with 23 species of bacteria and four species of fungus cultured from lesions that were suspected of contributing to local infection, systemic infection, or both. The most abundant bacteria included Aeromonas spp., Clostridium spp., Vibrio spp., Enterococcus faecalis, and Pseudomonas spp. In six cases, the microorganism(s) cultured were considered to have been associated with disease. Mixed infections containing Aeromonas spp. and Vibrio spp.; Morganella morganii, Pasteurella multocida, and Serratia marcescens; and Actinomyces spp. and Peptostreptococcus spp. were associated with pneumonia or pleuritis, and Enterococcus faecalis was associated with a multisystemic infection in a neonate. Clostridium spp. was cultured from two animals with peritonitis and likely septicemia. The significance of many of the other isolates is uncertain because the samples were taken after death, and some of the species isolated may represent postmortem overgrowth. It is also difficult to fulfil Kochs postulates through experimental infection in marine mammals. Regardless, this information will assist clinicians working with dugongs to make treatment decisions and the baseline data on the prevalence of bacterial and fungal species is of value for monitoring coastal water habitat health and risks of zoonotic disease transmission.

Molecular analysis of the genera Hapalotrema Looss, 1899 and Learedius Price, 1934 (Digenea: Spirorchiidae) reveals potential cryptic species, with comments on the validity of the genus Learedius

Adult blood flukes of the genera Hapalotrema Looss, 1899 and Learedius Price, 1934 were collected from turtles off Queensland and the Hawaiian Islands. Specimens were identified as Hapalotrema pambanensis Mehrotra, 1973, H. synorchis Luhman, 1935, H. postorchis Rao, 1976 and Learedius learedi Price, 1934 on the basis of morphology. No major morphological differences were found between specimens from this study and previously published descriptions. DNA was also extracted and sequences obtained using custom spirorchiid-specific primers for the ITS2 and 28S rDNA regions, in order to confirm species identification and investigate phylogenetic relationships. Intraspecific genetic variation was generally low. However the ITS2 region of H. postorchis and to a lesser extent that of L. learedi showed considerable variation between specimens from the Pacific and Atlantic oceans. Further studies will be required to determine whether this variation should be considered inter- or intra-specific. Maximum likelihood phylogenetic analyses were completed for both sequenced genes. Learedius learedi was unequivocally nested among species of Hapalotrema, suggesting that the status of the genus Learedius may need to be reassessed.

Trends in Marine Turtle Strandings along the East Queensland, Australia Coast, between 1996 and 2013

In-water monitoring of marine vertebrates is usually expensive while the use of stranding data can be used to provide a cost-effective estimation of disease and mortality. Strandings for Queensland are recorded in a web based database (StrandNet) managed by the Queensland Government’s Department of Environment and Heritage Protection (EHP). Data recorded in StrandNet from the east coast of Queensland between 1996 and 2013 were investigated for patterns of stranding. Significant trends in Queensland over this time were (i) an increase in the number of animals reported stranded within this study site; (ii) a species (loggerhead and green marine turtles) prevalence; (iii) a seasonal effect on different age classes stranding with most overall strandings occurring between August and November; and (iv) stranding hotspots (Moreton Bay, Hervey Bay, Rockhampton region, and Cleveland Bays) persisting throughout the study timeframe. This study suggested that intervention strategies, such as rehabilitation, should be able to be focussed on periods of heightened importance and specific localities to minimize health risks and contribute to sustainable use of resources.

Molecular Characterization of Coccidia Associated with an Epizootic in Green Sea Turtles ( Chelonia mydas ) in South East Queensland, Australia

In the spring of 2014, mass mortalities among wild green sea turtles occurred off the coast of south-east Queensland, Australia. The suspected causative agent was Caryospora cheloniae, an eimeriid coccidian implicated in previous epizootics. Necropsies were undertaken on a subset of 11 dead turtles, with subsequent histopathology and molecular analyses. All turtles returned positive PCR results for coccidial infection in various tissues; these included the brain, gastrointestinal tract, lung, kidney and thyroid. Granulomatous encephalitis was consistently observed, as well as enteritis and, less frequently, thyroiditis and nephritis. Sequencing and phylogenetic analyses indicated the presence of two distinct coccidian genotypes, presumably separate species—one associated with the brain, gastrointestinal tract and lung, and the second with the thyroid and kidney. Maximum likelihood and Bayesian inference analyses placed the first genotype closest to the lankesterellid genus Schellackia, rather than in the Eimeriidae, while the second was paraphyletic to the eimeriids. Presence of coccidial stages in extra-intestinal tissues of the primary host raises questions about the potential presence of intermediate or paratenic hosts within the life cycles, as well as their current placement relative to the genus Caryospora. This study represents the first genetic characterization of this emerging disease agent in green sea turtles, an endangered species, and has relevance for life-cycle elucidation and future development of diagnostics.

Ultrastructural and Molecular Characterisation of an Heterosporis-Like Microsporidian in Australian Sea Snakes (Hydrophiinae).

Four sea snakes (two Hydrophis major, one Hydrophis platurus, one Hydrophis elegans) were found washed ashore on different beaches in the Sunshine Coast region and Fraser Island in Queensland, Australia between 2007–2013. Each snake had multiple granulomas and locally extensive regions of pallor evident in the hypaxial and intercostal musculature along the body. Lesions in two individuals were also associated with vertebral and rib fractures. Histological examination revealed granulomas scattered throughout skeletal muscle, subcutaneous adipose tissue and fractured bone. These were composed of dense aggregates of microsporidian spores surrounded by a mantle of macrophages. Sequences (ssrRNA) were obtained from lesions in three sea snakes and all revealed 99% similarity with Heterosporis anguillarum from the Japanese eel (Anguillarum japonica). However, ultrastructural characteristics of the organism were not consistent with those of previous descriptions. Electron microscopic examination of skeletal muscle revealed large cysts (not xenomas) bound by walls of fibrillar material (Heterosporis-like sporophorocyst walls were not detected). The cysts contained numerous mature microsporidian spores arranged in small clusters, sometimes apparently within sporophorous vesicles. The microspores were monomorphic, oval and measured 2.5–3.0 μm by 1.6–1.8 μm. They contained isofilar polar filaments with 11 (infrequently 9–12) coils arranged in two ranks. This is the first published report of a microsporidian infection in hydrophiid sea snakes. This discovery shows microsporidia with molecular affinities to Heterosporis anguillarum but ultrastructural characters most consistent with the genus Pleistophora (but no hitherto described species). Further studies are required to determine whether the microsporidian presented here belongs to the genus Heterosporis, or to a polymorphic species group as suggested by the recognition of a robust Pleistophora/Heterosporis clade by molecular studies. The gross and histological pathology associated with these infections are described.

Establishment of reference intervals for plasma protein electrophoresis in Indo-Pacific green sea turtles, Chelonia mydas

Plasma protein electrophoresis analysis is now being used more frequently to improve our ability to diagnose disease in endangered species like green sea turtles. We developed plasma protein reference ranges using bloods from 55 health green sea turtles and used these ranges to diagnose disease syndromes in sick turtles.