Labeled quantitative mass spectrometry to study the host response during aspergillosis in the common bottlenose dolphin (Tursiops truncatus).

Aspergillosis is a fungal infection caused by Aspergillus molds that can affect both humans and animals. Despite advances in diagnostics and therapy, medical management of this disease remains difficult. Expansion of the basic knowledge regarding its pathophysiology in animals is critical to aid in the identification of new biomarkers of infection for diagnosis and therapeutic targets. For such a purpose, proteomics can be used by addressing protein changes during various disease processes. In the present study, a mass spectrometry analysis based on isobaric tagging for relative and absolute quantitation (iTRAQ®) was applied for direct identification and relative quantitation of proteins in blood collected from 32 Aspergillus-diseased common bottlenose dolphins (Tursiops truncatus, 32 samples) in comparison with blood from 55 other dolphins (55 samples from 41 clinically-normal controls and from 14 cetaceans with miscellaneous non-Aspergillus inflammation diseases) and ten convalescent dolphins (28 samples). Sixty-six and 40 proteins were found to be ≥2.0-fold over- and underrepresented versus miscellaneous non-Aspergillus inflammatory dolphins, respectively, and most were confirmed vs. clinically-normal controls and convalescents. Many proteins which play a role in the adaptive immune response were identified, including MHC proteins and others involved in catalytic activity like the NADPH-ubiquinone oxido-reductases. Overall, iTRAQ® appears to be a convenient proteomic tool greatly suited for exploratory ex vivo studies focusing on pathophysiology. This technique should be considered as a preliminary step before validation of new diagnostic markers.

Seroprevalence Survey for Microsporidia in Common Bottlenose Dolphin ( Tursiops truncatus): Example of a Quantitative Approach Based on Immunoblotting.

Little is known about microsporidiosis pathogenicity in cetaceans. Here we report seroprevalence of 76% for microsporidia in blood samples from common bottlenose dolphins ( Tursiops truncatus), from animals managed under human care ( n=108) or captured for health assessments ( n=13) and released.

Evaluation of a genus-specific ELISA and a commercial Aspergillus Western blot IgG® immunoblot kit for the diagnosis of aspergillosis in common bottlenose dolphins (Tursiops truncatus).

Aspergillosis is a fungal infection with high mortality and morbidity rates. As in humans, its definitive diagnosis is difficult in animals, and thus new laboratory tools are required to overcome the diagnostic limitations due to low specificity and lack of standardization. In this study of common bottlenose dolphins (Tursiops truncatus), we evaluated the diagnostic performance of a new commercial immunoblot kit that had been initially developed for the serologic diagnosis of chronic aspergillosis in humans. Using this in a quantitative approach, we first established its positive cutoff within an observation cohort of 32 serum samples from dolphins with "proven" or "probable" diagnosis of aspergillosis and 55 negative controls. A novel enzyme-linked immunosorbent assay (ELISA) test was also developed for detecting anti-Aspergillus antibodies, and results were compared between the two assays. Overall, the diagnostic performance of immunoblot and ELISA were strongly correlated (P < .0001). The former showed lower sensitivity (65.6% versus 90.6%), but higher specificity (92.7% vs. 69.1%), with no cross-reaction with other fungal infections caused by miscellaneous non-Aspergillus genera. When assessing their use in a validation cohort, the immunoblot kit and the ELISA enabled positive diagnosis before mycological cultures in 42.9% and 33.3% subjects addressed for suspicion of aspergillosis, respectively. There was also significant impact of antifungal treatment on the results of the two tests (P < .05). In all, these new serological methods show promise in aiding in the diagnosis of aspergillosis in dolphins, and illustrate the opportunity to adapt commercial reagents directed for human diagnostics to detect similar changes in other animals.

SARCOCYSTIS NEURONA-ASSOCIATED MENINGOENCEPHALITIS IN A PACIFIC WALRUS ( ODOBENDUS ROSMARUS DIVERGENS).

A 21-yr-old intact male walrus ( Odobendus rosmarus divergens) presented with acute onset of shifting lameness, initially associated with breeding behaviors. Further clinical signs manifested, including muscle tremors, anorexia, hematuria, and coughing. Diagnostics were limited, as the animal would not offer behaviors for voluntary sample collection. Signs were addressed with anti-inflammatories, anticonvulsants, and antibiotics. The walrus developed cluster seizures and ultimately, respiratory and cardiac arrest. Postmortem lesions included meningoencephalitis with intra- and extracellular protozoal zoites and schizonts, as well as interstitial pneumonia with intraendothelial protozoa. Immunolabeling of the protozoal organisms revealed Sarcocystis neurona. Previous S. neurona infections in an odobenid have not been reported. Protozoal infection should be considered in all species of captive marine mammals with nonspecific orthopedic, neurological, and respiratory clinical signs.

Ectopic pregnancy with associated gestational choriocarcinoma in a California sea lion (Zalophus californianus).

A wild-born, captive-reared, 14 yr old, primiparous female California sea lion Zalophus californianus presented for anorexia of 14 d duration and abdominal distention. Routine complete blood cell count revealed leukocytosis with a neutrophilia, and serum chemistry revealed hypoalbumenemia and hyponatremia. Treatment with broad spectrum antibiotics and non-steroidal anti-inflammatories were started, but the animal continued to decline. Abdominal radiographs revealed a mature mineralized fetal skull and spine in the caudal abdomen and abdominal ultrasound revealed ascites but could not confirm the fetus. The patient was taken to surgery where a full term fetus was found outside of the uterus but within the fetal membranes, representing a secondary ectopic pregnancy. The patient passed away during surgery and was taken to necropsy. Gross necropsy revealed a diffuse peritonitis with yellow deposits over the serosal surfaces of the abdominal organs. The uterus appeared intact grossly and the ovaries appeared abnormal. The mesenteric, renal, and sub-lumbar nodes were enlarged and edematous. Histopathology revealed choriocarcinoma in the right uterine horn with evidence of chronic uterine rupture and protrusion of the placental tissue into the abdomen. The choriocarcinoma had metastasized locally as well as to the liver, spleen and lung. Choriocarcinoma is a highly malignant trophoblastic neoplasm that is rare in domestic animals. This case represents, to the authors' knowledge, the first report of gestational choriocarcinoma causing secondary ectopic pregnancy in a California sea lion and presents questions regarding pregnancy monitoring and management in a population of captive, minimally trained California sea lions.

INTRAPERITONEAL DEXTROSE ADMINISTRATION AS AN ALTERNATIVE EMERGENCY TREATMENT FOR HYPOGLYCEMIC YEARLING CALIFORNIA SEA LIONS (ZALOPHUS CALIFORNIANUS).

The Marine Mammal Center (TMMC) cares for malnourished California sea lion (CSL) (Zalophus californianus) pups and yearlings every year. Hypoglycemia is a common consequence of malnutrition in young CSLs. Administering dextrose during a hypoglycemic crisis is vital to recovery. Traditional veterinary approaches to treat hypoglycemia pose therapeutic challenges in otariids, as vascular access and catheter maintenance can be difficult. The current approach to a hypoglycemic episode at TMMC is to administer dextrose intravenously (i.v.) by medically trained personnel. Intraperitoneal (i.p.) dextrose administration is an attractive alternative to i.v. administration because volunteer staff with basic training can administer treatment instead of waiting for trained staff to treat. This study compares the effects of i.v., i.p., and no dextrose administration on serum glucose and insulin in clinically healthy, euglycemic CSL yearlings. Three groups of animals, consisting of five sea lions each, were treated with 500 mg/kg dextrose using one of the following routes: i.v., i.p., or no dextrose (control). A jugular catheter was placed, and blood samples were collected at times 0, 5, 15, 30, 60, 120, 180, and 240 min after dextrose administration. I.v. dextrose administration resulted in an increase of serum glucose concentrations from a baseline level of approximately 150 mg/dl to a peak of approximately 350 mg/dl. The resulting hyperglycemia persisted for approximately 2 hr and was associated with an attenuated plasma insulin response compared with most terrestrial mammals. Intraperitoneal dextrose administration resulted in increases of serum glucose to approximately 200 mg/dl, which gradually declined to baseline by 2 hr after dextrose administration. These data suggest that the initial treatment of a hypoglycemic crisis in young malnourished CSLs can be accomplished with i.p. dextrose, thus enabling minimally trained volunteer staff to respond immediately to a crisis. Further studies are needed to determine the most appropriate long-term treatment.

Algal toxin impairs sea lion memory and hippocampal connectivity, with implications for strandings.

Domoic acid (DA) is a naturally occurring neurotoxin known to harm marine animals. DA-producing algal blooms are increasing in size and frequency. Although chronic exposure is known to produce brain lesions, the influence of DA toxicosis on behavior in wild animals is unknown. We showed, in a large sample of wild sea lions, that spatial memory deficits are predicted by the extent of right dorsal hippocampal lesions related to natural exposure to DA and that exposure also disrupts hippocampal-thalamic brain networks. Because sea lions are dynamic foragers that rely on flexible navigation, impaired spatial memory may affect survival in the wild.

Inflation and deflation pressure-volume loops in anesthetized pinnipeds confirms compliant chest and lungs.

We examined structural properties of the marine mammal respiratory system, and tested Scholander's hypothesis that the chest is highly compliant by measuring the mechanical properties of the respiratory system in five species of pinniped under anesthesia (Pacific harbor seal, Phoca vitulina; northern elephant seal, Mirounga angustirostris; northern fur seal Callorhinus ursinus; California sea lion, Zalophus californianus; and Steller sea lion, Eumetopias jubatus). We found that the chest wall compliance (CCW) of all five species was greater than lung compliance (airways and alveoli, CL) as predicted by Scholander, which suggests that the chest provides little protection against alveolar collapse or lung squeeze. We also found that specific respiratory compliance was significantly greater in wild animals than in animals raised in an aquatic facility. While differences in ages between the two groups may affect this incidental finding, it is also possible that lung conditioning in free-living animals may increase pulmonary compliance and reduce the risk of lung squeeze during diving. Overall, our data indicate that compliance of excised pinniped lungs provide a good estimate of total respiratory compliance.

Otariodibacter oris and Bisgaardia genomospecies 1 isolated from infections in pinnipeds.

We document the first associations of two recently described species of Pasteurellaceae bacteria with lesions in wild pinnipeds in rehabilitation. Samples were collected from nine lesions in four California sea lions (Zalophus californianus) and two Pacific harbor seals (Phoca vitulina) during necropsy or admission examinations at a rehabilitation facility in northern California. Seven Pasteurellaceae isolates were identified using phenotypic tests and partial rpoB gene sequencing. Six strains of Otariodibacter oris were isolated from California sea lions. Otariodibacter oris was isolated in pure culture from four abscesses, an affected lymph node, and a bone lesion consistent with osteomyelitis. Otariodibacter oris was also cultured with Arcanobacterium phocae and ß-hemolytic streptococci. A pure culture of Bisgaardia genomospecies 1 was obtained from an abscess in a harbor seal. This is the first time, to our knowledge, that O. oris has been associated with infection. Isolation of these bacteria in pure culture from abscesses and osteomyelitis strongly indicates a pathogenic potential of this organism. Likewise, the isolation of Bisgaardia genomospecies 1 in pure culture from an abscess in a harbor seal implies causality.