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1 ISSN , Volume 7, Number 1 This article was published in the above mentioned Springer issue. The material, including all portions thereof, is protected by copyright; all rights are held exclusively by Springer Science + Business Media. The material is for personal use only; commercial use is not permitted. Unauthorized reproduction, transfer and/or use may be a violation of criminal as well as civil law.

2 EcoHealth 7, , 2010 DOI: /s Ó 2010 International Association for Ecology and Health Original Contribution Health Surveillance of Stranded Green Turtles in Southern Queensland, Australia ( ): An Epidemiological Analysis of Causes of Disease and Mortality Mark Flint, 1 Janet C. Patterson-Kane, 2 Colin J. Limpus, 3 and Paul C. Mills 1 1 Veterinary-Marine Animal Research, Teaching and Investigation (Vet-MARTI) Unit, School of Veterinary Science, The University of Queensland, Therapies Road, St. Lucia, QLD 4072, Australia 2 Faculty of Veterinary Medicine, University of Glasgow, Bearsden Road, Glasgow G61 1QH, UK 3 Queensland Department of Environment, Resources and Management, George Street, Brisbane, QLD 4002, Australia Abstract: Causes of disease and mortality in marine turtles are frequently based on opportunistic investigations producing results that may not contribute to knowledge on how to protect their survival rate. Over a 4-year period ( ), the major causes of stranding and morbidity in 100 green turtles (Chelonia mydas) from southern Queensland on the east coast of Australia were determined by comprehensive postmortem examination. Lesions were characterized for analysis using descriptive and probability statistics. Spirorchiid parasitism was found to be the most frequently occurring cause of mortality (41.8%), followed by gastrointestinal impaction (11.8%), microbiological infectious diseases (5.2%), and trauma (5.2%). Spirorchiid parasitism with associated inflammation (75%) was the most frequently occurring disease, followed by gastrointestinal impaction (5.1%). All other diseases were observed at a low prevalence. Assessment of the likelihood of disease being influenced by risk factors (season, maturity, and gender) showed that: (i) there were more observed cases of spirorchiid infection in summer when compared with the other seasons (P = 0.029); (ii) immature turtles had more severe spirorchiid parasite infections than mature turtles (P = 0.032); and (iii) respiratory disorders were more likely (P = 0.01) in summer and autumn than winter or spring. Number of observed cases and severity of spirorchiid lesions were highest in the brain compared with other histologically examined organ systems (all P > 0.1). Further investigation is required to build on these findings, aid management decisions, and determine the significance of these diseases for green turtle survivorship in Queensland. Keywords: Chelonia mydas, green turtle, disease epidemiology, neurospirorchidiasis, marine, postmortem examination INTRODUCTION In recent decades, global fluctuations in green turtle (Chelonia mydas) population numbers have occurred Published online: March 16, 2010 Correspondence to: Mark Flint, m.flint@uq.edu.au (Chaloupka et al., 2008a), preserving their status in Australia as threatened under Federal (Environmental Protection and Biodiversity Conservation Act 1999) and State (Queensland s Nature Conservation Act 1992) legislations. The survivorship of marine turtle populations is important because they have been proposed as sentinel indicators of

3 136 Mark Flint et al. the health of their marine environments, many of which are used by human populations (Aguirre and Lutz, 2004). Direct and indirect anthropogenic factors are generally recognized as having the major impact on marine turtle population survivorship (Chaloupka et al., 2008b; Hamann et al., 2007; Harvell et al., 1999; Limpus, 2009a, b; Limpus and Limpus, 2003; Limpus et al., 2003). Environmental changes, particularly alterations in water temperature and resource availability, may be contributing to more frequent reports of emerging diseases, host shifts, and new disease manifestations (Harvell et al., 1999; Ward and Lafferty, 2004). For example, the debilitating disease fibropapillomatosis (FP), in which tumors develop in external and internal soft tissues in association with chelonian herpes virus infections (Greenblatt et al., 2005; Jacobson et al., 1991; Lu et al., 2000; Quackenbush et al., 2001; Stacy et al., 2008; Work et al., 2009), was first identified in green turtles in Florida in 1938 (Smith and Coates, 1938). However, this disease emerged as a global epidemic during the 1980s, affecting all marine turtle species except the leatherback (Dermochelys coriacea) (Aguirre and Lutz, 2004). Further manifestations, including corneal involvement, were not reported until at least the 1990s (Flint et al., 2009a; Jacobson et al., 1991). Causes of death of marine turtles in many regions of the world are unknown, thereby complicating their management. Until disease types are clearly documented, their frequencies and ranges of severity in the various species, and age and gender cohort predilection in different areas and seasons, including their clinical relevance and pathogenesis, the disease environment host relationship cannot be examined in any meaningful way. Currently, health and disease information is obtained from free-ranging necropsy studies (Gordon et al., 1993; Jacobson et al., 2006), extrapolated from captive management studies (Glazebrook and Campbell, 1990; Jacobson et al., 1979), reports of isolated natural disease outbreaks (Gordon et al., 1993; Greer et al., 2003; Jacobson et al., 2006), and short communications of previously undescribed diseases for a species or locality (Flint et al., 2009a; Jacobson et al., 1991; Limpus et al., 2009a, b). These reports are all that are available to rehabilitators to guide them when diagnosing and treating animals surrendered to their facilities. We determined the major causes of stranding and morbidity in 100 green turtles from southern Queensland by investigating the frequency of clinical signs, and the type, duration, severity, and etiology of both gross and histological lesions in animals that underwent postmortem examinations. We tested for any significant associations (i) between common antemortem clinical signs and common postmortem pathological diagnoses, (ii) between gross and microscopic diagnoses, and (iii) of disease with season, age, and gender. Further, we assessed the significance of the relationships of the number of observed cases and severity of lesions caused by spirorchiid parasites (iv) with season, age, and gender, and (v) between each examined organ system (cardiovascular, central nervous, gastrointestinal, and lymphoid). METHODS Turtles One hundred green turtles were obtained from southern Queensland (an area potentially extending to all embayments of the Sunshine Coast [25.88 S, E] in the north, Moreton Bay [27.20 S, E] centrally, and the Gold Coast [27.95 S, E] in the south between 2006 and Selection of turtles was restricted to animals that were accessible without causing environmental damage, were of suitable carcass condition to allow histological samples to be collected and interpreted, and/or where a diagnosis was required by interested parties. This equated to approximately 10% of the total number of green turtles reported stranded each year in this region being examined (12 animals up to 2006, 21 in 2007, 46 in 2008, and 21 in 2009, with an average of 255 green turtle strandings within the study area per year) (Greenland and Limpus, 2008a, b; Greenland et al., 2004). Government, rehabilitation, and tertiary institutes collaborated to collect these turtles. Of the green turtles presented to rehabilitation centers during this time, 75 (67 live and 8 dead) were submitted for postmortem examination based on the presumptive diagnosis that disease other than trauma alone was the cause of morbidity or death. Of these, 26 were given a guarded prognosis and euthanized without attempted rehabilitation, 41 died or were euthanized after attempted rehabilitation (minimum duration 1 day, maximum 300 days, average 29.1 days), and 8 presented dead. A further 25 green turtles (5 live and 20 dead) were submitted for postmortem examination to university veterinarians by government environmental officers following their routine patrols of the study area. Euthanasia was achieved by an intravenous overdose (100 mg/kg) of sodium pentobarbitone (Plumb, 1991) into the cervical dorsal sinus.

4 Disease and Mortality of Marine Turtles 137 When a carcass was not collected, a presumptive diagnosis was recorded in the Queensland Department of Environment and Resource Management s Stranding Network (STRANDNET) database, but not included in this study. If a carcass could not be collected from the wild within 24 hours of initial notification or from the refrigeration unit of the rehabilitation centers within 4 days, it was not included. Animals submitted to rehabilitation centers with traumatic injuries were likely to be successfully treated and released, so only 11 turtles with trauma (directly from the wild) were presented. These were used to provide baseline information (i.e., background pathology) on the functionally healthy component of the population, and to confirm that criteria used to determine disease were appropriate. Antemortem Examinations When presented with an antemortem patient (n = 72), a clinical examination was conducted in accordance with previously published guidelines to determine health status (Flint et al., 2009b). Disease was defined as absence of health determined by confirmation of one or more of the assessment criteria: abnormalities (fractures, trauma, infectious processes, high external parasite burdens, buoyancy disorders, and poor body condition), neurological deficits (atypical behaviors such as circling or nonresponsiveness), or evidence of FP (Chrisman et al., 1997; Flint et al., 2009b; Herbst and Jacobson, 2003; Work and Balazs, 1999). Poor body condition included cachexia, which was defined grossly as those animals with a sunken concave plastron that was easy to depress, and with obvious loss of muscle mass and/or fat around the limbs, neck, and eye sockets (Flint et al., 2009b). Postmortem Examinations Disease status was confirmed by postmortem examination. Comprehensive examinations included external assessment, recording of morphometric data, and internal examination of all major organ systems. Disease was defined and characterized by the detection of lesions in one or more organ. Carcasses were opened using a standard ventral approach with the animal placed in dorsal recumbency for full excision of the plastron to enable assessment and removal of each organ (Flint et al., 2009c, d; Work, 2000). For each turtle, standard representative tissues were collected (Flint et al., 2009c). Histological Processing and Examination Tissue specimens were processed routinely for histological examination after a minimum of 48 h fixation. The brain was fixed for at least 5 days before serial gross sectioning and fixation of those sections for a further 48 h. All specimens were embedded in paraffin wax, sectioned at 5 lm, and stained with hematoxylin and eosin (HE). Special stains including Ziehl-Neelsen (for acid-fast bacteria), Perl s Prussian blue (for iron pigment), and periodic acid- Schiff (for fungal/yeast elements) were used when indicated, following examination of routinely stained sections (Flint et al., 2009c). All sections were examined by an experienced specialist veterinary pathologist, and final morphological diagnoses were made based on both gross and histological diagnoses. Where lesions associated with spirorchiid trematode parasite infection (predominantly eggs with occasional adult worms) were noted (69 cases), the severity was subjectively scored in the cardiovascular (heart), central nervous (brain), gastrointestinal (gut), and lymphoid (spleen) systems as 0 (absent), 1 ( mild ), 2 ( moderate ), or 3 ( severe ). Histologically, mild lesions were defined as <1 to several small, scattered granulomas per low power (920) field, each centered on <5 trematode eggs. Moderate lesions were defined as 1 to several granulomas centered on >5 10 trematode eggs per 920 field, with some disruption of normal architecture. Severe lesions were defined as either (i) numerous smaller granulomas each centered on 5 eggs, or (ii) large, often coalescing granulomas and/or sheets of inflammatory cells centered on >10 to hundreds of eggs, both with extensive disruption of normal architecture. For lesions in the heart and intestine, the overall grade included the severity (i.e., extent) of lesions noted in those organs on gross examination, i.e., thrombosis and vasculitis (associated with adult parasites and/or eggs), and serosal granulomas (centered on eggs), respectively. Statistical Analysis For each examined turtle, clinical signs were categorized as source (received from a rehabilitation center or the wild), status (alert, moribund, or dead), body condition (poor, fair, or good), neurological (depressed, circling, or normal), and buoyancy (floating or normal). For each examined organ (bone, brain, heart, gastrointestinal tract, liver, lung, kidney, salt gland, spleen, and skeletal muscle), tissues were assessed grossly and histologically

5 138 Mark Flint et al. for process (infectious or inflammatory), duration (acute or chronic), severity of spirorchiid lesions (none, mild, moderate, or severe), and etiology (suspected diagnoses). Diagnoses were assigned based on interpretation of the ante- and postmortem results. The effect of source on final diagnosis was compared using t-tests in an Excel spreadsheet (Microsoft Office 2003 Excel, Microsoft Corp, Washington, DC). Data was grouped for subsequent analyses. Number of observed cases of each category for ante- and postmortem findings were determined; and number of observed cases and proportions of diseases and causes of death were calculated using Pivot Tables and descriptive statistics in an Excel spreadsheet. Each of these categories were grouped, where needed, to create a dichotomous result and assigned a numeric value (0 or 1) for pair-wise comparison. Pair-wise comparisons were calculated using Stata Statistical Software: Release 10 (2007, StataCorp, College Station, TX) to produce Chi-square tables with Fisher s exact t-test, Odds Ratios, and associated 95% confidence intervals for all anteand postmortem findings. For each turtle, risk factors of season (autumn, spring, summer, winter), age (immature and mature), and gender (female and male) were determined using a multivariable logistic regression model with variables fitted in the same model as fixed effects to produce results expressed as Fisher s exact t test, odds ratios, and associated 95% confidence intervals. The associations of the severity of spirorchiid parasiteassociated lesions (as graded on the basis of gross and/or histological lesions) with (i) season, age, and gender, and (ii) between cardiovascular, neurological, gastrointestinal systems, and the spleen were compared by Chi-square (two-way) tables producing proportion, and Fisher s exact test using Stata Statistical Software: Release 10. RESULTS Data Source and Population Structure Source Diagnoses of turtles sourced from the wild (n = 36) did not differ from those sourced from rehabilitation centers (n =64; P = 0.19), including if the turtle had been held for rehabilitation for more than 30 days (n = 8; P = 0.11). Irrespective of source, data was pooled for analyses. Population Structure For this study, examinations were conducted on 19 mature (a curved carapace length, CCL, of >90 cm), 11 large immature (CCL cm) and 59 small immature (CCL < 65 cm) turtles, with an additional 11 animals of unknown maturity. These included 33 males, 50 females, and 17 animals that were unsexed. The distribution of maturity and gender examined in this study were representative of the reported population structure for Moreton Bay (Limpus et al., 1994), except for a bias of 19% of turtles with a CCL > 90 cm versus 11% in the demographic study. Identification and Description of Causes of Mortality and Diseases Causes of Mortality The most frequently occurring causes of death were endoparasites, gastrointestinal disorders, generalized infection, and trauma, of which neurospirorchiidiasis and gastrointestinal and multiorgan spirorchiidiasis were the most frequent diagnoses (Table 1). Other individual causes of death were recorded at low rates, but the gastrointestinal and respiratory systems were frequently involved. Disease considered to contribute to cause of death was diagnosed in 92.8% (n =142) of examined green turtles, with 7.2% (n =11) of deaths assigned to anthropogenic misadventure, such as trauma or asphyxiation. Diseases Fifteen diseases affecting 12 systems diagnosed in the examined green turtles are given in Table 2. Irrespective of the system, spirorchiidiasis was the predominant disease with cardiovascular, central nervous system, gastrointestinal, splenic, respiratory, renal and salt gland granulomas centered on parasite eggs. Gastrointestinal impaction and cachexia also frequently occurred among the examined turtles. Not all the diagnosed diseases were considered to be the cause of death; for example, corneal FP, mild lesions caused by spirorchiids/eggs in all organs, some cachexia, renal oxalate crystal deposition, resolved carapace and limb trauma, hepatic lipidosis, and penile prolapse were considered to be incidental findings.

6 Disease and Mortality of Marine Turtles 139 Table 1. Causes of Mortality for Green Turtles Necropsied in Southern Queensland ( ) by Organ System, Irrespective of Season, Age, or Gender Disease n % of all diseases General Systemic microbiological infection Systemic inflammation Hypovolemia Trauma Spirorchiidiasis Neurological Cardiovascular Gastrointestinal Multiorgan Protozoal Unidentified apicomplexan parasite Coccidiosis Integumentary Fibropapillomatosis Digestive (gastrointestinal) Impaction Foreign body Inflammation (primary and secondary) Rupture Respiratory Asphyxiation Pneumonia (pulmonary inflammation) Neurological Meningoencephalitis (nonparasite) Excretory Nephritis Uro-cloacal-genital Penile prolapse Rectal abscessation Ectopic oviduct Skeletal Humeral osteitis Total 100 Range and Severity of Pathology The range and severity of pathology was described for common diseases. Spirorchiidiasis Adult spirorchiid trematode parasites and/or their eggs, the latter associated with granulomatous inflammation, were the most common disease occurring in 75% of examined turtles, often in multiple organs in the same animal, and contributed to death in 41.8% of examined cases. Spirorchiid eggs were found at varying frequency and severity in all examined systems, except the sensory, skeletal, and reproductive. Granulomas associated with eggs were most easily observable grossly in the intestinal serosa. Variable numbers of eggs within granulomas (heterophilic or histiocytic; egg granulomas ) were noted in intravascular or more usually perivascular locations; in the intestinal wall, these were most severe in the serosa and outer muscularis externa. Some egg-associated granulomas extended through vascular walls. Most egg granulomas were noted in the absence of any adult parasites, with the exception of lesions in the meninges and parenchyma of the brain, a few turtles with more severe intestinal inflammation, and grossly observable thrombi obstructing lumina of aortae or other major arteries. Adults were only noted grossly within aortic lesions; those in blood vessels in the brain and intestinal wall were microscopic. Intravascular parasites in the brain were associated with intramural and perivascular infiltrates of lymphoid cells, including cases where egg granulomas were not present. Lesions in the brain parenchyma were largely mild to moderate. Within the meningeal compartment, they were usually more severe and, in some cases, diffuse. Neurological deficits were only noted antemortem in turtles with severe neurospirorchiidiasis. Egg-associated granulomas within perivascular lymphoid tissue in the spleen were often associated with muscular hypertrophy of arterial vessel walls. In a few turtles, arterial muscular hypertrophy was also or alternatively noted in other tissues, including the lung and intestinal wall. Impaction Digestive disorders included impaction caused by torsion, obstruction of dry fecal balls, shell fragments, or intestinal constrictions in 11.8% of reported diseases, contributing to 5.1% of deaths. Etiology was not indicated, but gastrointestinal impactions occurred with moderate-tosevere spirorchiidiasis in 60% of cases. Foreign bodies were an additional cause of disease in 2.3% and death in 4.6% of the examined turtles, and included fishing lines and hooks, which were not necessarily associated with gastrointestinal ruptures (2.6%). Plastic or other flotsams were not recorded as a cause of disease or mortality. Impaction caused congestion of the vasculature and lymphatics supplying the gut. Microscopic lesions included inflammation, edema of the intestinal wall, and mucosal erosion.

7 140 Mark Flint et al. Table 2. Causes of Disease for Green Turtles Necropsied in Southern Queensland ( ) by Organ System, Irrespective of Season, Age, or Gender System Disease n % of samples % of all diseases General Cachexia Bacteriosis Integumentary Skin Fibropapilloma Sensory Eye Trauma Corneal fibropapilloma Muscular Spirorchiidiasis Skeletal Limbs Osteitis Skull Trauma Carapace Trauma Digestive Gut Spirorchiidiasis Coccidia Impaction Foreign body Liver Spirorchiidiasis Hepatic lipidosis Respiratory Lungs Spirorchiidiasis Mycobacter Asphyxiation Cardiovascular Heart and vessels Spirorchiidiasis Excretory Bladder Spirorchiidiasis Salt glands Spirorchiidiasis Kidney Spirorchiidiasis Renal oxalate crystal deposition Immune Spleen Spirorchiidiasis Neurological Brain Spirorchiidiasis Trauma Reproductive Penis Prolapse Ovary Ectopic uteri Total 100 Coccidia Coccidial enteritis due to Caryospora cheloniae infection was noted at varying severities in the gastrointestinal tract in 1.2% of examined turtles, and was considered a cause of disease and at least a contributory factor in cause of death. Lesions associated with this infection were the same as those previously described whereby an outbreak resulted in mass mortality (Gordon et al., 1993).

8 Disease and Mortality of Marine Turtles 141 Fibropapillomatosis Fibropapilloma tumors caused disease in 1.6% of examined turtles and were only proposed as a cause of death in 0.7%, a reported prevalence much lower than other places around the world (Chaloupka et al., 2008c; Oros et al., 2005). Cutaneous and corneal fibropapillomas were as previously described (Flint et al., 2009a; Jacobson et al., 1989; Smith and Coates, 1938). Microbiological Infection Infections caused by microbes were commonly diagnosed histologically during this investigation, however, were only assigned to causing an independent disease syndrome (bacteriosis) in 0.7% of cases and causing death in 5.2% of cases. Infections were noted in all systems except the reproductive, and at a range of lesion severities, from mild inflammatory changes to space-occupying lesions with well-developed fibrous encapsulation. For respiratory lesions, mycobacteria were the presumptive causative agents in three turtles, as acidfast bacteria were noted within the granulomas. Fungal hyphae were identified in a further case. Other microbial species were not identified by culture. Pneumonia Pneumonia was noted to contribute to 7.2% of deaths; it was not assigned as a disease, but rather a process associated with spirorchiidiasis, microbial infection, or asphyxiation. In addition to the signs noted under each disease, pneumonia was seen at a range of severities and included edema of the pulmonary interstitium. Nondisease-Related Causes of Mortality Nondiseased animals dying because of anthropogenic misadventure (7.2%) included those that asphyxiated while submerged in fishing nets (n =3) or received traumatic injuries as a result of a boat-strike (n =8). For five of these animals dying from acute processes, microscopic examination of tissues showed that 80% (4/5; two by asphyxiation and three by acute carapace trauma) had low infection rates of spirorchiidiasis of the brain (n =1), heart (n =1), spleen (n =3), or gastrointestinal tract (n =1), or a moderate infection rate of spirorchiidiasis of the brain (n =2). The remaining animal (1/5; acutely fractured carapace) had no parasite lesions. Microscopic examination of tissues from one turtle with chronic severe head trauma showed signs of necrosis, osteoblastic activity of the skull, and rupture and calcification of an eye. This animal had a moderate infection rate of spirorchiidiasis of the brain, heart, and spleen, and a mild infection rate of spirorchiidiasis of the gastrointestinal tract. Risk Factors (Season, Age, and Gender) Increasing Likelihood of Disease Number of observed cases for any of the measured diagnoses did not vary between seasons, age, or gender. The exception was the histologic examination of lung tissues, which showed respiratory lesions were more likely (P = 0.01) to be observed during summer (89%, 8/9) or autumn (43%, 6/14) than in winter (25%, 2/8) or spring (29%, 7/24). Based on the histologic examination of 69 turtles, spirorchiids were found in 88% (53/60) of examined spleens, 84% (47/56) of brains, 55% (36/66) of gastrointestinal tracts, and 54% (37/69) of heart samples. Analysis of these samples, collected from a range of animals throughout the year, showed that season, and turtle age and gender, had a limited relationship with the number of observed cases and severity of spirorchiid infection (P > 0.05). Exceptions were season, where the severity of observed cases with spirorchiid lesions in the gastrointestinal tract were higher (35%; 15/43) in the warmer than cooler seasons (26%; 6/23) (P = 0.029); and age, where only immature turtles were observed to have severe spirorchiid lesions (P = 0.032). Relationships of Clinical Signs, Findings, and Diagnoses There was no correlation between presenting clinical signs (mentation, body condition, neurological status, and buoyancy), gross and histologic findings (process, duration, severity, and etiology), and final diagnoses, except for neurological deficits (n =4) that were always associated with severe grade histological lesions (two due to parasites, one to trauma, and one to microbial infection) of the brain. Of interest, buoyancy disorders were not correlated with gross (P = 0.22 [OR = 1.9 (95% CI = )]) or histologic (0.78 [1.1 ( )]) gastrointestinal lesions, or with gross (0.19 [2.0 ( )]) and histologic (0.40 [0.8 ( )]) respiratory lesions. For all examined comparisons of selected common gross and histologic tissues, only the severity of gastrointestinal lesions were significantly correlated (0.004 [7.4 ( )]). Similarity of Spirorchiidiasis Between Systems The number of observed cases and the severity of spirorchiid lesions in the brain did not correlate with that for the

9 142 Mark Flint et al. heart, gastrointestinal tract, and spleen (P = 0.185, 0.175, and 0.237, respectively). By contrast, the number of observed cases and the severity of spirorchiid lesions for the latter three organs were similar to one another (all P < 0.005). DISCUSSION Disease is speculated to be a considerable contributing factor to marine animal mortality (Deem et al., 2001), but not all global populations are subject to the same disease processes or frequencies. In this Moreton Bay study, spirorchiidiasis was by far the most common cause of disease and mortality among the green turtles examined; this exceeded figures reported for this region from over a decade before (Gordon et al., 1998), which suggested an increase in the pathogenic effect of this disease over this time. Its effect in other parts of the world, Hawaii for example, has been reported at variable frequencies (Chaloupka et al., 2008c; Work et al., 2005). In contrast, FP, a disease causing significant devastation in the United States, had a very minor influence on disease or death in the current study (Chaloupka et al., 2008c). The subset of animals included in this study where cause of death was due to anthropogenic misadventure had low-grade infection rates of spirorchiids in multiple organs, with moderate-grade infections seen in the brain of a few. Little is known about the lifecycle and pathology of spirorchiid parasites in marine turtles (Flint et al., 2009c), so it was impossible to tell if these infections increased the likelihood of anthropogenic misadventure by enhancing risk-taking behavior (a phenomenon reported in other species, such as rodents infected with Toxoplasma gondii [Holliman, 1997]), or whether these infection rates were incidental findings. Until these parasites lifecycle(s) and pathogeneses can be determined, it can be assumed that low-grade infections are incidental within the healthy population. Difficulty in obtaining apparently healthy animals for this study may have biased the frequency, organ distribution, and range of severity of specific diseases identified. Presenting clinical signs did not correlate with final diagnoses. This was not surprising due to the subtle nature of clinical signs in reptiles, and variation in the experience of clinicians, making largely subjective observations that may not have an underlying scientific basis. This is likely to result in incorrect prescription of treatment regimes and a reduced likelihood of successful rehabilitation. Buoyancy disorders are frequently diagnosed and treated based on speculation that gastrointestinal and respiratory conditions have caused entrapment of gas in those respective systems (Wyneken et al., 2006). The results of this study indicated that such diseases had no greater association with a buoyancy disorder than any other examined disease. Additionally, in this study, there was a disappointingly poor association between gross and microscopic diagnoses with the exception of spirorchiidiasis, possibly as it was easily identified grossly in organs due to formation of surface- and vascular-associated egg granulomas. Otherwise, as for any species, diagnoses were likely to be missed if based on gross examination only, which has been common practice for marine turtles (Flint et al., 2009c; Terrell and Stacy, 2007). Comprehensive collection and examination of histological samples (Flint et al., 2009c) should therefore be standard. It could alternatively be argued that microscopic examination elevated the reported frequency of clinical disease above what is actually causing impediment to the population. Diseases requiring microscopic diagnosis may not affect a system to the level whereby function is impeded, thus not contributing to morbidity or mortality (Jacobson et al., 2006). Unfortunately, there is no comparative information on the presence or absence, severity or organ distribution, of such lesions in turtles that are clinically healthy, including those associated with spirorchiid infection, other than those presented in this study. Spirorchiidiasis has been reported previously as a common cause of morbidity and mortality of green turtles in southern Queensland waters (Glazebrook et al., 1989; Gordon et al., 1998; Raidal et al., 1998) and other parts of the world (Jacobson et al., 2006; Santoro et al., 2007; Work et al., 2005). However, this is the first study to document gross and histological lesions of all severities, investigate seasonal differences in the number of observed cases and severity of lesions, and determine differences between organ systems. The increase in severity of gastrointestinal spirorchiidiasis during warmer seasons may be due to enhanced cercarial emergence, or an increased availability of the intermediate host(s), such as marine snails (Flint et al., 2009c). It is also possible that warmer temperatures stimulate greater egg production in worms already resident in turtles. Overall predominance of gastrointestinal disorders in the warmer seasons may also correlate to spirorchiidiasis, as associated inflammatory lesions were most severe in outer layers of the intestinal wall, where they were

10 Disease and Mortality of Marine Turtles 143 frequently noted to impinge on or replace myenteric ganglia, and to be associated with vascular damage (the latter as in other sites). These lesions may have predisposed turtles to intestinal accidents (i.e., torsion) and impactions. Additionally, chronic gastrointestinal illnesses managed during winter may increase in clinical significance during warmer seasons when turtles are subjected to nutrient-depleted food resources, increased human activities (usage of waterways and fishing practices), increased activity (energetically costly migration or mating in mature animals), or other pathogens, such as Caryospora cheloniae (Chaloupka et al., 2008c; Gordon et al., 1993). Reasons for immature turtles having the entire range of severity for spirorchiid lesions of the gastrointestinal tract, when mature turtles only displayed mild lesions, could include greater exposure to intermediate hosts as immature turtles enter embayments, or immature turtles being immunologically naive when they enter the embayment. It is also possible that naturally immunologically resistant turtles maintain only low parasite infection throughout life, irrespective of exposure levels, while susceptible turtles or those that accidentally swim through a cloud of cercariae develop heavy infection at a young age, dying before they reach maturity. The number of observed cases of parasite lesions (i.e., their eggs and associated inflammatory responses) for each of the cardiovascular, lymphoid, and gastrointestinal systems did not correlate with that for the central nervous system. This may be due to anatomical reasons (i.e., where certain adult parasites penetrate the body and where they lodge within the cardiovascular system), or the brain may be preferentially infected by different parasite species to those that infect other organ systems. The latter hypothesis was supported by the frequent observation of microscopic adult parasites (up to 8 mm in length), within meningeal and parenchymal vessels in inflamed brain tissue, that were significantly smaller than adults (approximately 12 mm in length) identified within aortic thrombi. If parasites in the brain have the greatest clinical significance (Jacobson et al., 2006), and if the species are separate, there is a need for an antemortem test that identifies the presence of spirorchiids causing neurological deficits. Little information is known regarding the causes of disease and mortality in marine turtles. This study has extended diagnoses beyond clinical findings to include comprehensive gross and histologic examination. Furthermore, we determined the relationships between pathologic data and potential risk factors, such as season and maturity, to guide future investigation of the pathogenesis of locally important diseases. ACKNOWLEDGMENTS The authors thank the indigenous people of Quandamooka who kindly allowed us access to turtles on their traditional lands and waters; Drs. Ristan Greer and John Morton (School of Veterinary Science, The University of Queensland) for advice and assistance with the statistical analyses; Prof. David Blair (School of Marine and Tropical Sciences, James Cook University) for constructive comments on the manuscript, particularly the parasitology component; and staff of Australia Zoo (Beerwah), Sea World (Gold Coast), Underwater World (Mooloolaba), and Queensland Parks and Wildlife (Cleveland) for procuring carcases. Samples were procured under Moreton Bay Marine Park Authority Permit QS2008/CVL1414 and Scientific Purposes Permit WITK with The University of Queensland animal ethics approval (SVS/624/08). This research was funded by grants from the Australian Government Department of the Environment, Water, Heritage and the Arts Marine Species Recovery and Protection Grants Programme, the Great Barrier Reef Marine Park Authority, and SEQ Healthy Waterways. REFERENCE Aguirre AA, Lutz P (2004) Sea turtles as sentinels of marine ecosystem health: is fibropapillomatosis an indicator? EcoHealth 1: Chaloupka M, Bjorndal KA, Balazs GH, Bolten AB, Ehrhart LM, Limpus CJ, et al. (2008a) Encouraging outlook for recovery of a once severely exploited marine megaherbivore. Global Ecology and Biogeography 17: Chaloupka M, Kamezaki N, Limpus CJ (2008b) Is climate change affecting the population dynamics of the endangered Pacific loggerhead sea turtle? Journal of Experimental Marine Biology and Ecology 356: Chaloupka M, Work T, Balazs G, Murakawa S, Morris R (2008c) Cause-specific temporal and spatial trends in green sea turtle strandings in the Hawaiian archipelago ( ). Marine Biology 154: Chrisman CL, Walsh M, Meeks JC, Zurawka H, LaRock R, Herbst LH (1997) Neurological examination of sea turtles. Journal of American Veterinary Medical Association 211: Deem SL, Karesh WB, Weisman W (2001) Putting theory into practice: wildlife health in conservation. Conservation Biology 15: Flint M, Limpus CJ, Patterson-Kane JC, Murray PJ, Mills PC (2009a) Corneal fibropapillomatosis in green sea turtles (Che-

11 144 Mark Flint et al. lonia mydas) in Australia. Journal of Comparative Pathology. doi: /j.jcpa Flint M, Morton JM, Limpus CJ, Patterson-Kane JC, Murray PJ, Mills PC (2009b) Development and application of biochemical and haematological reference intervals to identify unhealthy green sea turtles (Chelonia mydas). The Veterinary Journal. doi: /j.tvjl Flint M, Patterson-Kane JC, Limpus CJ, Work TM, Blair D, Mills PC (2009c) Post mortem diagnostic investigation of disease in free-ranging marine turtle populations: a review of common pathological findings and protocols. Journal of Veterinary Diagnostic Investigation 21: Flint M, Patterson-Kane JC, Mills PC, Limpus CJ (2009d) A veterinarian s guide to sea turtle post mortem examination and histological investigation. html?page= Glazebrook JS, Campbell RS, Blair D (1989) Studies on cardiovascular fluke (Digenea: Spirorchiidae) infections in sea turtles from the Great Barrier Reef, Queensland, Australia. Journal of Comparative Pathology 101: Glazebrook JS, Campbell RSF (1990) A survey of the diseases of marine turtles in northern Australia I. Farmed turtles. Disease of Aquatic Organisms 9:83 95 Gordon AN, Kelly WR, Cribb TH (1998) Lesions caused by cardiovascular flukes (Digenea: Spirorchidae) in stranded green turtles (Chelonia mydas). Journal of Veterinary Pathology 35:21 30 Gordon AN, Kelly WR, Lester RJ (1993) Epizootic mortality of free-living green turtles, Chelonia mydas, due to coccidiosis. Journal of Wildlife Diseases 29: Greenblatt RJ, Quackenbush SL, Casey RN, Rovnak J, Balazs GH, Work TM, et al. (2005) Genomic variation of the fibropapilloma-associated marine turtle herpesvirus across seven geographic areas and three host species. Journal of Virology 79: Greenland JA, Limpus CJ (2008a) III. Marine turtles. In: Queensland Marine Wildlife Stranding and Mortality Database Annual Report, 2004, Brisbane, Australia: Queensland Environmental Protection Agency, p 49 Greenland JA, Limpus CJ (2008b) III. Marine turtles. In: Queensland Marine Wildlife Stranding and Mortality Database Annual Report, 2003, Brisbane, Australia: Queensland Environmental Protection Agency, p 63. Greenland JA, Limpus CJ, Currie KJ (2004) III. Marine turtles. In: Queensland Marine Wildlife Stranding and Mortality Database Annual Report , Vol 3, Brisbane, Australia: Environmental Protection Agency, Queensland Parks Wildlife Service, p 73. Greer LL, Strandberg JD, Whitaker BR (2003) Mycobacterium chelonae osteoarthritis in a Kemp s Ridley sea turtle (Lepidochelys kempii). Journal of Wildlife Diseases 39: Hamann M, Limpus CJ, Read MA (2007) Vulnerability of marine reptiles in the Great Barrier Reef to climate change. In: Climate Change and the Great Barrier Reef: A Vulnerability Assessment, Johnson JE, Marshall PA (editors), Townsville, Australia: Great Barrier Reef Marine Parks Authority, pp Harvell CD, Kim K, Burkholder JM, Colwell RR, Epstein PR, Grimes DJ, et al. (1999) Emerging marine diseases climate links and anthropogenic factors. Science 285: Herbst LH, Jacobson ER (2003) Practical approaches for studying sea turtle health and disease. In: The Biology of Sea Turtles, Vol 2, Lutz P, Musick JA, Wyneken J (editors), New York: CRC Press, pp Holliman RE (1997) Toxoplasmosis, behaviour and personality. Journal of Infectious Diseases 35: Jacobson ER, Buergelt C, Williams B, Harris RK (1991) Herpesvirus in cutaneous fibropapillomas of the green turtle Chelonia mydas. Disease of Aquatic Organisms 12:1 6 Jacobson ER, Gaskin JM, Shields RP, White FH (1979) Mycotic pneumonia in mariculture-reared green sea turtles. Journal of American Veterinary Medical Association 175: Jacobson ER, Homer BL, Stacy BA, Greiner EC, Szabo NJ, Chrisman CL, et al. (2006) Neurological disease in wild loggerhead sea turtles Caretta caretta. Disease of Aquatic Organisms 70: Jacobson ER, Mansell JL, Sundberg JP, Hajjar L, Reichmann ME, Ehrhart LM, et al. (1989) Cutaneous fibropapillomas of green turtles (Chelonia mydas). Journal of Comparative Pathology 101:39 52 Limpus CJ (2009) A Biological Review of Australian Marine Turtles. 4. Olive Ridley Turtle, Lepidochelys olivacea (Eschscholtz), Brisbane, Australia: Queensland Environmental Protection Agency, pp 27 Limpus CJ (2009) A Biological Review of Australian Marine Turtles. 6. Leatherback Turtle, Dermochelys coriacea (Vandelli), Brisbane, Australia: Queensland Environmental Protection Agency, pp 29 Limpus CJ, Couper PJ, Read MA (1994) The green turtle, Chelonia mydas, in Queensland: population structure in a warm temperate feeding area. Memoirs of the Queensland Museum 35: Limpus CJ, Limpus DJ (2003) The biology of the loggerhead turtle, Caretta caretta in western, South Pacific Ocean foraging areas. In: Biology and Conservation of Logerhead Turtles, Witherington B, Bolten A (editors), Washington, DC: Smithsonian Institution Press, pp Limpus CJ, Limpus DJ, Read MA, FitzSimmons NN (2009) When is a male turtle not a male? Observations on intersex turtles Chelonian Conservation and Biology 8: Limpus CJ, Miller JD, Limpus DJ (2009) The occurrence of ectopic cloaca deformity in the green turtle in eastern Australia. Chelonian Conservation and Biology 8: Limpus CJ, Miller JD, Paramenter CJ, Limpus DJ (2003) The green turtle, Chelonia mydas, population of Raine Island and the northern Great Barrier Reef: Memoirs of the Queensland Museum 49: Lu Y, Wang Y, Yu Q, Aguirre AA, Balazs GH, Nerurkar VR, et al. (2000) Detection of herpesviral sequences in tissues of green turtles with fibropapilloma by polymerase chain reaction. Archives of Virology 145: Oros J, Torrent A, Calabuig P, Deniz S (2005) Diseases and causes of mortality among sea turtles stranded in the Canary Islands, Spain ( ). Diseases of Aquatic Organisms 63:13 24 Plumb DC (1991) Veterinary Drug Handbook Pocket Edition, White Bear Lake, MN: PharmaVet, pp Quackenbush SL, Casey RN, Murcek RJ, Paul TA, Work TM, Limpus CJ, et al. (2001) Quantitative analysis of herpesvirus sequences from normal tissue and fibropapillomas of marine turtles with real-time PCR. Virology 287: Raidal SR, Ohara M, Hobbs RP, Prince RI (1998) Gram-negative bacterial infections and cardiovascular parasitism in green sea

12 Disease and Mortality of Marine Turtles 145 turtles (Chelonia mydas). Australian Veterinary Journal 76: Santoro M, Morales J, Rodríguez-Ortíz B (2007) Spirorchiidiosis (Digenea: Spirorchiidae) and lesions associated with parasites in Caribbean green turtles (Chelonia mydas). Veterinary Record 161: Smith GM, Coates CW (1938) Fibro-epithelial growths of the skin in large marine turtles, Chelonia mydas (Linnaeus). Zoologica (N.Y.) 23:93 98 Stacy BA, Wellehan JF, Foley AM, Coberley SS, Herbst LH, Manire CA, et al. (2008) Two herpesviruses associated with disease in wild Atlantic loggerhead sea turtles (Caretta caretta). Veterinary Microbiology 126:63 73 Terrell SP, Stacy BA (2007) Reptile necropsy techniques. In: Infectious Diseases and Pathology of Reptiles: Color Atlas and Text, Jacobson ER (editor), Boca Raton, FL: CRC Press, pp Ward JR, Lafferty KD (2004) The elusive baseline of marine disease: are diseases in ocean ecosystems increasing? PLoS Biology 2: Work TM (2000) Sea Turtle Necropsy Manual for Biologists in Remote Refuges, Honolulu: United States Geological Survey Work TM, Balazs GH (1999) Relating tumor score to hematology in green turtles with fibropapillomatosis in Hawaii. Journal of Wildlife Diseases 35: Work TM, Balazs GH, Schumacher JL, Amarisa M (2005) Epizootiology of spirorchiid infection in green turtles (Chelonia mydas) in Hawaii. Journal of Parasitology 91: Work TM, Dagenais J, Balazs G, Schumacher JL, Lewis TD, Leong JC, et al. (2009) In vitro biology of fibropapilloma-associated turtle herpesvirus and host cells in Hawaiian green turtles (Chelonia mydas). Journal of General Virology 90: Wyneken J, Mader DR, Weber ES, Merigo C (2006) Medical care of sea turtles. In: Reptile Medicine Surgery, Mader DR (editor), 2nd ed. St, Louis: Saunders Elsevier, pp