MEC&F Expert Engineers : ADRENAL GLAND AND LUNG LESIONS IN GULF OF MEXICO COMMON BOTTLENOSE DOLPHINS (TURSIOPS TRUNCATUS) FOUND DEAD FOLLOWING THE DEEPWATER HORIZON OIL SPILL

Thursday, May 21, 2015

ADRENAL GLAND AND LUNG LESIONS IN GULF OF MEXICO COMMON BOTTLENOSE DOLPHINS (TURSIOPS TRUNCATUS) FOUND DEAD FOLLOWING THE DEEPWATER HORIZON OIL SPILL








MAY 20, 2015

Abstract

A northern Gulf of Mexico (GoM) cetacean unusual mortality event (UME) involving primarily bottlenose dolphins (Tursiops truncatus) in Louisiana, Mississippi, and Alabama began in February 2010 and continued into 2014. 

Overlapping in time and space with this UME was the Deepwater Horizon (DWH) oil spill, which was proposed as a contributing cause of adrenal disease, lung disease, and poor health in live dolphins examined during 2011 in Barataria Bay, Louisiana. 

To assess potential contributing factors and causes of deaths for stranded UME dolphins from June 2010 through December 2012, lung and adrenal gland tissues were histologically evaluated from 46 fresh dead non-perinatal carcasses that stranded in Louisiana (including 22 from Barataria Bay), Mississippi, and Alabama. 

UME dolphins were tested for evidence of biotoxicosis, morbillivirus infection, and brucellosis. Results were compared to up to 106 fresh dead stranded dolphins from outside the UME area or prior to the DWH spill. 

UME dolphins were more likely to have primary bacterial pneumonia (22% compared to 2% in non-UME dolphins, P = .003) and thin adrenal cortices (33% compared to 7% in non-UME dolphins, P = .003). In 70% of UME dolphins with primary bacterial pneumonia, the condition either caused or contributed significantly to death. 

Brucellosis and morbillivirus infections were detected in 7% and 11% of UME dolphins, respectively, and biotoxin levels were low or below the detection limit, indicating that these were not primary causes of the current UME. The rare, life-threatening, and chronic adrenal gland and lung diseases identified in stranded UME dolphins are consistent with exposure to petroleum compounds as seen in other mammals. 

Exposure of dolphins to elevated petroleum compounds present in coastal GoM waters during and after the DWH oil spill is proposed as a cause of adrenal and lung disease and as a contributor to increased dolphin deaths.


Introduction

A large, multi-year cetacean unusual mortality event (UME) has been ongoing in the northern Gulf of Mexico (GoM) since February 2010, continuing into 2014 [1]. This event has involved predominantly (87%) common bottlenose dolphins (Tursiops truncatus) (hereafter referred to as ‘dolphins’) stranded in Louisiana, Mississippi, and Alabama [2]. 

The UME coincided with the Deepwater Horizon (DWH) oil spill, the largest marine-based spill in U.S. history [3]. During and following the DWH oil spill, significantly elevated polycyclic aromatic hydrocarbon (PAH) levels attributed to this spill were detected in coastal GoM waters, including Louisiana, Mississippi, and Alabama [4]. These locations coincided with the states most impacted by the ongoing UME since the DWH oil spill [2]. 

Dolphin strandings, however, were elevated during March and April before the spill, necessitating an investigative approach including numerous potential causes [1,2,5]. Combined oil exposure, an unusually cold winter during 2011, and fresh water infusions have been proposed as potential causes contributing to this UME [6].

Barataria Bay, Louisiana was one of the heaviest oiled coastal areas from the DWH oil spill, including visualized oiling from the spill encompassing 40 km and 366,000 m2 of Barataria Bay’s shoreline lasting in decreasing amounts for at least 2 years [710]. The presence of increased coastal PAH levels associated with the DWH oil spill, especially near Grand Isle, Louisiana in Barataria Bay have been confirmed [4]. 

Further, within the time period of January 2010 to June 2013, the longest lasting cluster of dolphin strandings throughout the northern GoM was in Barataria Bay (August 2010 through 2011) [2]. During the DWH oil spill and response period, numerous dolphins, including dolphins in Barataria Bay, were observed swimming through visibly oiled waters and feeding in areas of surface, subsurface, and sediment oiling [11].

Due to the extensive oiling in Barataria Bay, health assessments were conducted on live dolphins in this area during the summer of 2011 [11]. Barataria Bay dolphins had a high prevalence of moderate to severe lung disease and blood value changes indicative of hypoadrenocorticism; specific blood changes included low serum cortisol, aldosterone, and glucose, and high neutrophil counts [11]. 

Nearly half (48%) of Barataria Bay dolphins were given a guarded to grave prognosis for long-term survival [11]. The DWH oil spill was proposed as a contributor to adrenal gland and lung disease in live Barataria Bay dolphins.

Previous to the ongoing event, there have been ten dolphin GoM UMEs since 1991, as well as one large die-off during 1990 that occurred before the UME declaration process [1, 1215]. The majority (82%) of previous dolphin GoM events had brevetoxicosis or morbillivirus as confirmed or suspected causes [1].

 While brevetoxicosis events do not leave a histologic signature in affected dolphins, brevetoxicosis-related events are often associated with known algal blooms and deaths that appear to be acute in otherwise healthy-looking dolphins [15]. In prior events classified as brevetoxicosis-related, 50% or more sampled dolphins were positive for brevetoxin with most at high concentrations [15]. 

Similarly, past UMEs that have been attributed to morbillivirus involved successful detection of morbillivirus in greater than 60% of dolphins tested. [13,16]. There is evidence that Brucella, which is commonly found in marine mammals worldwide, can cause disease in cetaceans, including bottlenose dolphins [1721]. 

As such, there was a need to evaluate all of these potentially important diseases as playing contributing or leading roles in the ongoing UME.
To assess contributing factors and causes of deaths for stranded UME dolphins following the DWH oil spill, tissues were histologically evaluated from 46 carcasses that stranded in Louisiana, Mississippi, and Alabama, including 22 from Barataria Bay, from June 2010 through December 2012. 

Perinatal dolphins, stranded dolphins that were less than 115 cm in body length that likely died during late-term pregnancy or shortly after birth, were excluded from this study. On the basis of the live dolphin health assessment findings from Barataria Bay, this study included a focused evaluation of adrenal, lung, and liver lesions with the expectation that if stranded dolphins had been impacted by the DWH oil, they would have lesions consistent with the clinical evidence indicating lung disease and hypoadrenocorticism found in live dolphins. 

Other potential causes of and contributors to dolphin deaths were investigated, including the presence of histologic lesions and diagnostic test results consistent with brevetoxicosis, morbillivirus infections, and brucellosis. Results were compared to a reference group of fresh dead dolphins from North Carolina, South Carolina, Texas, and the Gulf coast of Florida that stranded prior to or remote from the UME and DWH oil spill timeframes and geographic location.

Discussion
To our knowledge, adrenal cortical atrophy as found in this study has not been previously described in free-ranging cetaceans, including bottlenose dolphins previously studied in the northern GoM [32]. The normal corticomedullary ratio of dolphin adrenal glands has been determined to be approximately 1:1 [24].

 Thus, the discovered high prevalence of adrenal cortical atrophy in dolphins stranding during the ongoing GoM UME may be part of a syndrome that has not been previously reported in dolphins during mortality events. 

The prevalence of adrenal cortical atrophy identified in this study is consistent with the high prevalence (approximately 50%) of live Barataria Bay dolphins with evidence of hypoadrenocorticism assessed during 2011, including a relatively high proportion of dolphins with low blood cortisol, aldosterone, and glucose [11]. Follow up evaluation of adrenal glands from stranded dolphins in subsequent years will help to determine the persistence of adrenal insufficiency observed relative to the timing of the UME and the concurrent DWH oil spill.


There are a number of different causes of adrenal insufficiency in mammals, including autoimmune disease, metastatic neoplasia, fungal infections, stress, trauma, miliary tuberculosis, corticosteroid toxicity, and contaminant exposure [33]. Additionally, infection with phocine herpesvirus-1 has been demonstrated to cause adrenal cortical necrosis in marine mammals [34]. 

In the current study, only 2 of 46 UME dolphins had inflammation in the adrenal gland, and with the exception of one case with a disseminated bacterial infection, neither infectious agents nor neoplasia were identified in UME dolphin adrenal glands. Further, there was no histologic evidence of autoimmune adrenalitis or neoplasia in any UME dolphin adrenal glands, indicating that adrenal cortical atrophy in UME dolphins was not due to direct infection of the adrenal gland, autoimmune disease, or neoplasia.

In humans, chronic demand on the adrenal glands, including chronic illness, has been postulated to lead to cortical thinning and potential adrenal exhaustion associated with lipid depletion of the fasciculata cells [35,36]. Previous evaluations of adrenal glands from stranded GoM dolphins from Texas with both acute and chronic disease have been conducted, but no cases of adrenal cortical atrophy were identified [32]. 

Instead, adrenal glands of dolphins dying from chronic disease (likely chronically stressed individuals) were significantly heavier, and corticomedullary ratios were significantly higher than those dying from acute disease or acute trauma. 

Findings from Clark et al. (2006) suggest that adrenal gland enlargement and cortical hyperplasia are common responses to chronic stress and disease in bottlenose dolphins, similar to that noted in other cetaceans and other mammalian species [24, 32, 3739]. 

Of the 12 UME dolphins with a thin adrenal gland cortex, one-third had primary bacterial pneumonia, leaving the majority of adrenal cortex cases without evidence of active or chronic infections. Further, none of the UME dolphins with a thin adrenal gland cortex had depleted cardiac adipose tissue, indicating that UME dolphins were not in an advanced, debilitated nutritional state. These results do not support general infection or chronic poor body condition as underlying causes of adrenal gland cortex depletion.

Although the effects of polycyclic aromatic hydrocarbon (PAHs) on the hypothalamus-pituitary-adrenal (HPA) axis are poorly understood, the adrenal gland is reported to be the most common endocrine organ to exhibit lesions with exposure to toxigenic chemicals [40, 41].

 In general, mechanisms of direct adrenal toxicity include impaired steroidogenesis, activation of toxins by cytochrome p450 enzymes generating reactive oxygen metabolites, DNA damage, and exogenous steroid action [42]. The adrenal gland can be a significant site for metabolism of PAHs, thus increasing the adrenal gland to exposure from these contaminants and their metabolites [43].

Several studies have shown that PAHs or oil can affect the HPA axis and adrenal gland function. Hypoadrenocorticism has been reported in mink fed either bunker C or artificially weathered fuel oil [44,45]. In these mink, adrenal cortical hypertrophy with vacuolation of corticocytes was detected histologically. 

These studies, however, did not monitor changes in response to higher level exposure and/or over longer periods of time. Chemicals that induce adrenal cortical vacuolar degeneration can lead to loss of adrenocortical cells due to necrosis and adrenal cortical atrophy. 

It is possible PAHs may act in a similar fashion [42]. Naphthalene, a common PAH associated with crude oil, reduced plasma corticosterone in mallard ducks following ingestion of petroleum-contaminated food, and a similar acute decrease in cortisol was detected in exposed eels [46, 47]. 

House sparrows exposed orally to 1% crude oil from the GoM exhibited decreases in cortisol in response to stressors or to adrenocorticotropin hormone injection [48].

Mammalian exposure to PAHs can greatly increase hepatic metabolism of other compounds (e.g. 7,12-dimethylbenz(α)anthracene), which in turn can cause targeted and severe injury to the adrenal gland, including necrosis and hemorrhage [4951]. 

Removal of the inciting chemical, if the adrenal cortical injury is not too advanced, may result in regained function and resolved lesions characterized by fibrosis, atrophy, nodular regeneration or calcification [42, 47, 51]. 

Ultrastructural analysis can be beneficial in helping to identify direct toxic damage. Unfortunately, optimally fixed, minimally autolyzed tissue from affected dolphin adrenal glands was not available for ultrastructural analysis. The lack of adrenal lesions beyond cortical atrophy suggests, however, that potential chemical effects may be higher in the HPA axis [52].

During and following the DWH oil spill, significantly elevated PAH levels were detected in the coastal GoM waters, including Louisiana, Mississippi, and Alabama [53]. These locations coincide with the states most impacted by the ongoing UME since the DWH oil spill [1]. 

Thus, northern GoM dolphins’ exposures to DWH spill-associated PAHs, especially in Louisiana and Mississippi, may account for the observed effects on adrenal function found in both live and dead dolphins [11]. Given the lack of evidence of alternative causes of adrenal cortical atrophy and the high prevalence of this lesion among stranded dolphins following the DWH oil spill, the leading hypothesis is that exposure to contaminants from the DWH oil spill led to chronic injury of the adrenal gland cortex at least through 2012.

Chronic adrenal insufficiency (CAI) is a life-threatening disease that can lead to adrenal crisis and death in mammals [53]. Adrenal crises in people with CAI are triggered by infections, fever, major pain, psychological distress, heat, and pregnancy [54]. Cold temperatures can also increase the risk of death among animals with CAI. 

Angora goats with a genetically-driven high incidence of primary CAI lack proper cortisol and glucose response and, as such, are susceptible to die-offs from cold stress [54, 55]. GoM dolphins were exposed to colder than normal temperatures during early 2011, and if those dolphins from the UME had pre-existing CAI, they may have been at higher risk of cold stress-related deaths [6]. 

This hypothesis is further supported in that dolphins have a compensatory adrenal response in cold temperatures, including increased cortisol levels, presumably to help generate metabolic heat [56]. 

Adrenal crisis may have been the cause of death for many of the UME dolphins with adrenal cortical atrophy following stress events to which a healthy dolphin could have otherwise adapted. In addition to the cold weather during 2011, adrenal crisis could have also been precipitated by late-term pregnancies and infections, including bacterial pneumonia [57].

Compared to reference dolphins, UME dolphins were more likely to have a primary bacterial pneumonia. Many of these pneumonias were much more severe than bacterial pneumonias in the reference dolphins. These findings are consistent with the high prevalence of moderate to severe lung disease detected in live Barataria Bay dolphins [11]. 

During the DWH oil spill and response period, numerous dolphins, including dolphins in Barataria Bay, were observed swimming through visibly oiled waters and feeding in areas of surface, subsurface, and sediment oiling [11]. As mentioned, the presence of increased coastal PAH levels associated with the DWH oil spill, especially near Grand Isle, Louisiana in Barataria Bay, have been confirmed, indicating an increased risk of inhaled PAHs in dolphins [4]. 

Given that the dolphin's blowhole is at the surface of the water, chemicals, including volatile PAHs, could have been readily inhaled. In other animals, inhaled PAHs can irritate airways, denude mucosal surfaces, and cause peribronchial inflammation and systemic toxicity [58]. Damaged epithelium and cilia, in turn, can severely impair immune defenses.

In other animals, the severity of chemical inhalation injury is dependent on breathing patterns, in which deep breaths increase injury to tissues deeper within the lung [59]. This pattern is of particular importance given the dolphin's respiratory anatomy and physiology. 

While humans exchange approximately 10 to 20% of air with each breath, dolphins exchange 75 to 90% of deep lung air [6063]. They also lack nasal turbinates and cilia to filter the air prior to reaching the lungs, and have deep inhalations followed by a breath hold that provides potential for more prolonged contact and exchange between air-borne particulates and the blood [6063]. All of these factors would likely amplify the effects of inhaled chemical irritants in dolphins compared to observations and studies involving other mammals.

The severe bacterial pneumonias found in UME dolphins could represent a chronic sequelae to hydrocarbon inhalation or aspiration, or have been secondary to PAH induced immune system compromise. 

The most common sequela to hydrocarbon inhalation and ingestion in humans and animals are aspiration pneumonia and pneumonia often involving the bronchioles [6468]. Inhaled hydrocarbon vapors or aspirated hydrocarbons may cause necrosis of bronchial and bronchiolar epithelium, and pneumocyte and alveolar septal necrosis which leads to inflammation and secondary infection [6466]. 

During the 2007 firestorm in San Diego, dolphins and people living in San Diego Bay area were exposed to high levels of PAHs [69, 70]. The month following the fires, these dolphins demonstrated decreased absolute and percent neutrophils [70]. This change indicated that dolphins exposed to PAHs through inhalation may have had a compromised immune response and an increased risk of acquiring bacterial pneumonia.

In addition to inhalation risks, hydrocarbon ingestion can lead to gastrointestinal mucosal irritation, vomiting or regurgitation, and resultant aspiration pneumonia. Cattle that ingest petroleum develop bacterial pneumonia due to chemical-induced regurgitation and/or aspiration [67,68]. 

Correspondingly, based on histologic examination, one dolphin that stranded during June 2010 in Mississippi during the DWH oil spill had suspected aspiration pneumonia, secondary bacterial infection, ulcerative tracheitis, and ulcerative gastritis with edema. Both the tracheal and gastric lesions, although non-specific, could have resulted from mucosal irritation, such as may occur with toxin ingestion.

Though there was no difference in prevalence of liver lesions when comparing UME and reference dolphins in this study, two UME dolphins had similar severe centrilobular liver lesions characterized by hepatocyte loss, necrosis and vacuolation that could potentially be associated with toxin exposure. 

Both of these dolphins stranded in Barataria Bay. Hepatocellular vacuolation, degeneration and necrosis have been associated with exposure to crude oil and benzo[a]pyrene (BaP) [71 72]. 

Hepatotoxic liver injury may occur due to xenobiotic metabolism of substances producing injurious metabolites and lesions most often occur in the centrilobular zones where hepatocytes have the highest concentration of cytochrome p450 enzymes [71]. 

Other rule-outs for centrilobular degeneration and necrosis include hypoxia, severe or precipitous anemia (e.g. hemolytic anemia), chronic heart disease, or circulatory failure associated with septic shock [72, 73]. 

There was no other evidence of hypoxia, hemolytic anemia or heart disease in either of the affected dolphins and lesions were more chronic than would be expected secondary to acute hypoxia or shock. Some oiled sea otters that died following the Exxon Valdez oil spill had centrilobular hepatic necrosis, though whether the lesions were due to direct toxic insult or secondary to anemia is unclear [74]. 

Biliary or periportal inflammation and fibrosis secondary to infection by the trematode Campula spp. are common hepatic lesions noted in a number of cetacean species, and periportal lesions noted in both UME and reference dolphins were consistent with the chronic sequelae of biliary trematode infection [75].

This study did not support that previously documented or suspected contributing factors for GoM dolphin UMEs were primary contributors to the ongoing UME among non-perinatal dolphins. All UME dolphins in this case study had biotoxin levels that were below detectable levels except for one with low levels [12]. 

Relatively few morbillivirus cases were identified among UME cases. In previous known dolphin morbillivirus-associated die-offs, more than 60% of cases tested positive for the virus when using a similar PCR assay [14,16]. 

Exposure to morbillivirus has been documented in GoM dolphins, and the cases identified in 2011 and 2012 may represent exposure to the virus in a small number of susceptible individuals in the population [76]. 

Similarly, there were too few brucellosis cases in this study to explain the ongoing UME, with only two cases that had Brucella identified in the lung, demonstrating that Brucella was not the driver for increased primary bacterial pneumonia. Despite global reports of Brucella infections in marine mammals, there have been, to date, no documented brucellosis epizootics in cetaceans [17].

Due to the long duration and large scope of the ongoing UME, there may be multiple factors affecting the health of dolphins by region through time. Aside from the DWH oil spill, there were two relatively smaller oil spills that occurred in and around Barataria Bay during this study’s timeframe. Specifically, the T/V Pere Ana C spill in Mud Lake, Louisiana on July 27, 2010 (approximately 7,000 gallons spilled) and the Cedyco Manilla Village Spill in Bayou Dupont, Louisiana which occurred on September 11, 2011 (approximately 10,500 gallons) [77,78].

 In comparison, however, the DHW oil spill released approximately 126,000,000 gallons and was visible across 40 km and 366,000 m2 of Barataria Bay’s shoreline in decreasing amounts over time for at least 2 years, demonstrating the higher magnitude of the DWH oil spill’s likely impact compared to other spills [710].

The Gulf of Mexico has historically had documented dead zones with episodes of seasonal hypoxia associated with nutrient loading from the Mississippi River watershed [79,80]. 

The geographic area and clusters of dolphin stranding identified from the current UME, however, were not limited to single seasons or specific dead zone hotspots [2].

Further, dead zones are often associated with fish die-offs and habitat loss; the lack of emaciation as the primary contributor to the deaths of dolphins in this study supports that the primary driver of this UME was not loss of prey [80].


The lack of baseline diagnostic and histologic data on fresh stranded dolphins prior to 2010 in the UME area, as well as during the pre-DWH oil spill period, paired with an assumption that stranded dolphins should have similar lesion prevalence regardless of location, are limitations in this study. 

The surprisingly high number of assessed lesions that were not significantly different between the UME and reference dolphins, however, increased the confidence that the study groups were indeed comparable. Continual assessment of trends and changing disease states over time are needed, however, to better understand the potential roles of multiple contributing factors to dolphin mortality during the ongoing UME.

In summary, UME dolphins had a high prevalence of thin adrenal gland cortices (especially in Barataria Bay dolphins) and primary bacterial pneumonia. These findings are consistent with endocrinologic and pulmonary-based observations of live bottlenose dolphins from health assessments in Barataria Bay during 2011 [11]. 

Previously documented or suspected contributing factors for GoM UMEs (marine biotoxins, morbillivirus, and brucellosis) were not supported by this study as contributors to the ongoing UME. Due to the timing and nature of the detected lesions, we hypothesize that contaminants from the DWH oil spill contributed to the high numbers of dolphin deaths within this oil spill’s footprint during the northern GoM UME following the DWH oil spill.

 Direct causes of death likely included: 1) affected adrenal gland cortices, causing chronic adrenal insufficiency, 2) increased susceptibility to life-threatening adrenal crises, especially when challenged with pregnancy, cold temperatures, and infections, and 3) increased susceptibility to primary bacterial pneumonia, possibly due to inhalation injury, aspiration of oil, or perturbations in immune function.

Source: http://journals.plos.org