Inga F. Sidor

The role of domoic acid in abortion and premature parturition of California sea lions (Zalophus californianus) on San Miguel Island, California.

Domoic acid is a glutaminergic neurotoxin produced by marine algae such as Pseudo-nitzschia australis. California sea lions (Zalophus californianus) ingest the toxin when foraging on planktivorous fish. Adult females comprise 60% of stranded animals admitted for rehabilitation due to acute domoic acid toxicosis and commonly suffer from reproductive failure, including abortions and premature live births. Domoic acid has been shown to cross the placenta exposing the fetus to the toxin. To determine whether domoic acid was playing a role in reproductive failure in sea lion rookeries, 67 aborted and live-born premature pups were sampled on San Miguel Island in 2005 and 2006 to investigate the causes for reproductive failure. Analyses included domoic acid, contaminant and infectious disease testing, and histologic examination. Pseudo-nitzschia spp. were present both in the environment and in sea lion feces, and domoic acid was detected in the sea lion feces and in 17% of pup samples tested. Histopathologic findings included systemic and localized inflammation and bacterial infections of amniotic origin, placental abruption, and brain edema. The primary lesion in five animals with measurable domoic acid concentrations was brain edema, a common finding and, in some cases, the only lesion observed in aborted premature pups born to domoic acid–intoxicated females in rehabilitation. Blubber organochlorine concentrations were lower than those measured previously in premature sea lion pups collected in the 1970s. While the etiology of abortion and premature parturition was varied in this study, these results suggest that domoic acid contributes to reproductive failure on California sea lion rookeries.

Partial Replacement of Fish Meal with Soy Protein Concentrate in Diets of Atlantic Cod

Abstract In two feeding trials conducted with juvenile Atlantic cod Gadus morhua (initial mean weight = 87.9 or 15.92 g), soy protein concentrate (SPC) was used to replace fish meal in isonitrogenous, isocaloric diets. In the first trial, five experimental diets (47% protein, 15% fat; calculated gross energy = 5,059 kcal/kg) were formulated to replace 28, 60, 80, and 100% of dietary fish meal. In the second trial, three diets were formulated with SPC to replace 0, 25, and 50% of the fish meal in a modified commercial diet for marine finfish (50% protein, 15% fat; calculated gross energy = 4,900 kcal/kg; calculated digestible energy = 3,570 kcal/kg). Upon conclusion of the studies, no differences in survival, growth, hepatosomatic index, or viscerosomatic index were found among the treatment groups. The fish attained mean weights of 162.7 and 39.1 g in the first and second trials, respectively, with specific growth rates of 0.76% and 1.12% and feed conversion ratios of 1.28 and 1.29, respectively. No consi...

Development, validation, and utilization of a competitive enzyme-linked immunosorbent assay for the detection of antibodies against Brucella species in marine mammals.

A competitive enzyme-linked immunosorbent assay (cELISA) was developed by using a whole-cell antigen from a marine Brucella sp. isolated from a harbor seal (Phoca vitulina). The assay was designed to screen sera from multiple marine mammal species for the presence of antibodies against marine-origin Brucella. Based on comparisons with culture-confirmed cases, specificity and sensitivity for cetacean samples tested were 73% and 100%, respectively. For pinniped samples, specificity and sensitivity values were 77% and 67%, respectively. Hawaiian monk seal (Monachus schauinslandi; n = 28) and bottlenose dolphin (Tursiops truncatus; n = 48) serum samples were tested, and the results were compared with several other assays designed to detect Brucella abortus antibodies. The comparison testing revealed the marine-origin cELISA to be more sensitive than the B. abortus tests by the detection of additional positive serum samples. The newly developed cELISA is an effective serologic method for detection of the presence of antibodies against marine-origin Brucella sp. in marine mammals.

HEMATOLOGIC AND PHYSIOLOGIC REFERENCE RANGES FOR FREE-RANGING ADULT AND YOUNG COMMON LOONS (GAVIA IMMER)

Abstract Wild common loons (Gavia immer) were captured across much of their southern North American range. Hematologic and physiologic data were collected to establish reference ranges for adults and chicks from four geographic areas: New England, Canadian Maritimes, south-central Quebec, and the Upper Great Lakes. Mean body mass and blood-glucose levels of adult loons varied among geographic regions. Chicks had lower PCV and total solids than adult loons, and also had faster heart rates and higher blood glucose levels. Although mean body mass in males was consistently greater than in females, hematologic and physiologic reference ranges between sexes were not significantly different. Geographic and age-related differences exist in common loon hematologic and physiologic reference ranges and should be considered when assessing such parameters.

Myofasciitis in the domestic ferret

Since late 2003, an inflammatory disease of muscle and fascia has been diagnosed in several ferrets at Northwest ZooPath, and this report describes the condition in 17 ferrets. It is a disease of young ferrets, characterized by rapid onset of clinical signs, high fever, neutrophilic leukocytosis, treatment failure, and death (or euthanasia). Gross lesions include atrophy of skeletal muscle; red and white mottling and dilatation of the esophagus; and splenomegaly. Histologically, moderate to severe suppurative to pyogranulomatous inflammation is in the skeletal muscle and the fascia at multiple sites, including esophagus, heart, limbs, body wall, head, and lumbar regions. Myeloid hyperplasia of spleen and/or bone marrow also is a prominent feature. Ultrastructural lesions include mitochondrial swelling, intracellular edema, disruption of myofibrils and Z bands. Bacterial and viral cultures, electron microscopy, immunohistochemistry, and polymerase chain reaction were negative for a variety of infectious agents. The clinical presentation and distribution of lesions suggests that polymyositis in domestic ferrets is likely a distinct entity. The etiopathogenesis if this condition is not known.

BRUCELLA PINNIPEDIALIS INFECTIONS IN PACIFIC HARBOR SEALS (PHOCA VITULINA RICHARDSI) FROM WASHINGTON STATE, USA

Abstract In 1994 a novel Brucella sp., later named B. pinnipedialis, was identified in stranded harbor seals (Phoca vitulina). This Brucella sp. is a potential zoonotic pathogen and is capable of causing disease in domestic animals. Serologic, microbiologic, and pathologic data collected from live captured and stranded harbor seals were used to better describe the epizootiology of B. pinnipedialis in harbor seals from Washington State, USA, in 1994 through 2006. We found no sex predilection in harbor seal exposure or infection with B. pinnipedialis but noted a significant difference in prevalence among age classes, with weaned pups, yearlings, and subadults having highest exposure and infection. The most common postmortem finding in 26 Brucella-positive animals (culture and/or PCR) was verminous pneumonia due to Parafilaroides spp. or Otostrongulus circumlitus. Our data are consistent with exposure to B. pinnipedialis post-weaning, and it is likely that fish or invertebrates and possibly lungworms are involved in the transmission to harbor seals. Brucella pinnipedialis was cultured or detected by PCR from seal salivary gland, lung, urinary bladder, and feces, suggesting that wildlife professionals working with live, infected seals could be exposed to the bacterium via exposure to oral secretions, urine, or feces. Endangered sympatric wildlife species could be exposed to B. pinnipedialis via predation on infected seals or through a common marine fish or invertebrate prey item involved in its transmission. More work is required to elucidate further potential fish or invertebrates that could be involved in the transmission of B. pinnipedialis to harbor seals and better understand the potential risk they could pose to humans or sympatric endangered species who also consume these prey items.

A multiplex real-time polymerase chain reaction assay with two internal controls for the detection of Brucella species in tissues, blood, and feces from marine mammals

Brucellosis has emerged as a disease of concern in marine mammals in the last 2 decades. Molecular detection techniques have the potential to address limitations of other methods for detecting infection with Brucella in these species. Presented herein is a real-time polymerase chain reaction (PCR) method targeting the Brucella genus–specific bcsp31 gene. The method also includes a target to a conserved region of the eukaryotic mitochondrial 16S ribosomal RNA gene to assess suitability of extracted DNA and a plasmid-based internal control to detect failure of PCR due to inhibition. This method was optimized and validated to detect Brucella spp. in multiple sample matrices, including fresh or frozen tissue, blood, and feces. The analytical limit of detection was low, with 95% amplification at 24 fg, or an estimated 7 bacterial genomic copies. When Brucella spp. were experimentally added to tissue or fecal homogenates, the assay detected an estimated 1–5 bacteria/µl. An experiment simulating tissue autolysis showed relative persistence of bacterial DNA compared to host mitochondrial DNA. When used to screen 1,658 field-collected marine mammal tissues in comparison to microbial culture, diagnostic sensitivity and specificity were 70.4% and 98.3%, respectively. In addition to amplification in fresh and frozen tissues, Brucella spp. were detected in feces and formalin-fixed, paraffin-embedded tissues from culture-positive animals. Results indicate the utility of this real-time PCR for the detection of Brucella spp. in marine species, which may have applications in surveillance or epidemiologic investigations.

Serologic response in bottlenose dolphins Tursiops truncatus infected with Brucella sp. using a dolphin-specific indirect ELISA

Marine-origin Brucella infections and serologic evidence of exposure have been documented in multiple cetacean species. A dolphin-specific indirect enzyme-linked immunosorbent assay (ELISA) was developed to screen bottlenose dolphin sera for anti-Brucella antibodies. A total of 131 serum samples collected over a 2 to 18 yr period from 6 bottlenose dolphins Tursiops truncatus with confirmed Brucella infections were analyzed for the presence and magnitude of antibody titers against marine-origin Brucella to compare individual antibody responses to various disease manifestations. Additionally, an epidemiologic serologic survey of a managed population of 64 bottlenose dolphins was performed to evaluate for the presence of antibodies and to determine whether there were any clinical pathology predictors for exposure or infection. The serologic results revealed that the dolphins with Brucella-associated abortions were seronegative for 7 to 18 yr until after the abortion and maintained positive titers for several years, with 2 of 3 animals returning to seronegative status. In contrast, the dolphins with Brucella-associated pulmonary or bone lesions maintained persistent positive titers for 2 to 18 yr. The population serosurvey revealed no significant differences in antibody levels among males and females, and dolphins between the ages of 17 and 25 yr were 6.8 times more likely to be Brucella antibody positive compared to those that were younger or older. Seropositive dolphins did not have significant inflammation compared to seronegative dolphins but were more likely to have higher levels of aspartate aminotransferase and gamma-glutamyl transpeptidase. Among 16 dolphins that tested seropositive, 13 (81.3%) had previously been seropositive for at least 3 to 5 yr.

Chronic pancreatitis with secondary diabetes mellitus treated by use of insulin in an adult California sea lion

CASE DESCRIPTION A 21-year-old neutered male captive California sea lion developed chronic polyuria; polydipsia; polyphagia; accelerated development of existing cataracts; and frequent episodes of gastrointestinal upset including anorexia, signs of abdominal discomfort, diarrhea, and vomiting. CLINICAL FINDINGS Chronic hypercholesterolemia, hypertriglyceridemia, hyperglycemia, and glucosuria were identified. During episodes of gastrointestinal abnormalities, transient hyperbilirubinemia and increased serum J-glutamyltransferase activities developed. Clinical findings strongly suggested chronic pancreatitis with secondary diabetes mellitus and intermittent cholestasis. Multiple diagnostic tests, including abdominal ultrasonography, serial hematologic and serum biochemical analyses, fecal examinations, urinalyses and bacteriologic culture of urine, measurement of serum fructosamine and insulin concentrations, and evaluation of thyroid and adrenal function, did not reveal any specific parasitic, endocrine, hepatic, or neoplastic etiologies. TREATMENT AND OUTCOME For 1.5 years, the sea lion received once-daily administration of glargine insulin, gastrointestinal protectants, and a strict high-protein, low-fat diet. Daily monitoring of glucose regulation was achieved by training the sea lion to submit to blood and urine sampling. Glucose regulation ranged from fair to good, and clinical signs of diabetes mellitus lessened. Episodes of gastrointestinal upset still occurred, although the frequency and severity decreased. Ultimately, a severe episode developed, associated with diabetic ketoacidosis and sepsis, and the sea lion died. Severe fibrosing pancreatitis with exocrine and endocrine atrophy and abscesses arising from ectatic pancreatic ducts were found. Peripancreatic fibrosis caused stricture of the common bile duct, resulting in gallbladder distension without cholecystitis. CLINICAL RELEVANCE Diabetes mellitus can occur secondary to chronic pancreatitis in California sea lions and insulin therapy should be considered.

Systemic Mycosis in a California Sea Lion (Zalophus californianus) with Detection of Cystofilobasidiales DNA

Abstract:  A 6-yr-old, intact male California sea lion (Zalophus californianus) with a systemic mycosis died after 5 wk of antifungal drug therapy. Antemortem clinical findings included hind flipper swelling, ring-lesions on skin of the flippers, and dermal nodules that increased in size and number spreading from the hind flippers and ventral abdomen to the foreflippers and muzzle. Lesions were accompanied by severe lymphadenopathy and development of systemic clinical signs despite therapy using itraconazole and later voriconazole. Histopathologic evaluation of biopsies revealed granulomatous dermatitis due to infection by fungus-producing yeast cells in tissue. Isolation attempts, using biopsied skin and tissue samples collected at necropsy, failed to yield growth of a fungus producing yeast cells like those in histologic section. Consensus polymerase chain reaction (PCR) tests of biopsied skin for fungal DNA produced an amplicon having significant sequence identity with a Cystofilobasidiales, a fungus belonging to a subclade that includes several Cryptococcus spp. Histopathologic evaluation of necropsy tissues revealed a systemic mycosis with yeast cells disseminated throughout subcutis, lymph nodes, and viscera. Hepatic necrosis was identified associated with acute liver failure, possibly from the voriconazole administration. This is the first report documenting the clinical presentation, treatment, and pathologic findings of infection associated with Cystofilobasidiales in a marine mammal and serves to expand the understanding of mycoses in pinnipeds.