Stephanie A. Norman

SEASONAL HEMATOLOGY AND SERUM CHEMISTRY OF WILD BELUGA WHALES (DELPHINAPTERUS LEUCAS) IN BRISTOL BAY, ALASKA, USA

We collected blood from 18 beluga whales (Delphinapterus leucas), live-captured in Bristol Bay, Alaska, USA, in May and September 2008, to establish baseline hematologic and serum chemistry values and to determine whether there were significant differences in hematologic values by sex, season, size/age, or time during the capture period. Whole blood was collected within an average of 19 min (range=11–30 min) after the net was set for capture, and for eight animals, blood collection was repeated in a later season after between 80–100 min; all blood was processed within 12 hr. Mean hematocrit, chloride, creatinine, total protein, albumin, and alkaline phosphatase were significantly lower in May than they were in September, whereas mean corpuscular hemoglobin concentration, monocytes, phosphorous, magnesium, blood urea nitrogen, alanine aminotransferase, aspartate aminotransferase, γ-glutamyltranspeptidase, and creatinine kinase were significantly higher. Mean total protein, white blood cell count, neutrophils, and lymphocytes were significantly higher early in the capture period than they were later. No significant differences in blood analyte values were noted between males and females. Using overall body length as a proxy for age, larger (older) belugas had lower white blood cell, lymphocyte, and eosinophil counts as well as lower sodium, potassium, and calcium levels but higher creatinine levels than smaller belugas. These data provide values for hematology and serum chemistry for comparisons with other wild belugas.

Maternal–Fetal Transmission of Cryptococcus gattii in Harbor Porpoise

To the Editor: We report maternal–fetal transmission of Cryptococcus gattii and death in a wild porpoise. Cryptococcus neoformans and C. gattii are 2 environmental, encapsulated yeasts that cause invasive, potentially life-threatening infections in humans and animals (1). C. neoformans causes disease in immunocompromised hosts, and C. gattii is also pathogenic in immunocompetent hosts (2). Since 1999, cryptococcosis caused by C. gattii has appeared on southern Vancouver Island (British Columbia, Canada) and nearby surrounding areas (2,3). Spread beyond Vancouver Island has been documented along the Pacific Northwest Coast, but the mechanism remains undetermined (4). A pregnant, dead, stranded, harbor porpoise (Phocoena phocoena) was reported on February 22, 2007, on western Whidbey Island, in Puget Sound, Washington State (48.2833°N, 122.7283°W). The carcass was iced and necropsy was performed on February 24. Sampled tissues from the adult and fetus were divided: half fixed in 10% formalin for histopathologic analysis, and half frozen for ancillary studies. For histologic analysis, tissues were embedded in paraffin, sectioned to 3–5 μm, and stained with hematoxylin and eosin. Selected sections were stained with mucicarmine. The adult porpoise (length 177 cm, weight ≈57.7 kg) was in poor condition (reduced blubber layer). Both lungs were exposed, extensively scavenged, firm, and nodular; a sectioned surface exuded clear to slightly opaque gelatinous to mucinous discharge. Mediastinal lymph nodes were grossly enlarged, multinodular, and firm with large numbers of yeasts visible by microscopy (online Figure A1 panel A, www.cdc.gov/EID/content/17/2/304-appF.htm). The first stomach chamber contained two 3.5 cm × 2.5 cm raised, centrally umbilicated ulcers and several embedded anisakid nematodes. The uterus was gravid in the right horn with a mid-term fetus. No other gross lesions were identified. Microscopically, the lung lesions correlated with granulomatous to pyogranulomatous infiltrates, often with a myriad of yeasts. The male fetus (length 30 cm, weight 2.4 kg), was examined separately at a different facility than the dam. It appeared grossly normal externally and was at a gestation of ≈5–6 months. Mediastinal lymph nodes had mild granulomatous inflammation and contained numerous yeasts morphologically consistent with Cryptococcus spp. (Figure A1, panel B). The lymph nodes were partially replaced with intracellular and extracellular mulilobulated yeast aggregates (length 8–20 μm) with pale eosinophilic central regions and a thin refractile wall peripherally bound by a 5-μm nonstaining capsule. Around the periphery of these aggregates, there were small numbers of macrophages and lymphocytes and fewer neutrophils. Specific staining showed a prominent mucicarminophilic capsule consistent with Cryptococcus spp. Yeasts were found in the amniotic fluid and interspersed within the chorioallantoic villi and submucosal vasculature of the placenta. Mild multifocal nonsuppurative myocarditis was detected. However, no yeasts were seen in inflamed areas. There were no overt lesions in the remaining organs. Maternal and fetal tissues were cultured for fungi, and diagnosis was based on Gram stain (budding yeast-like cells), India ink stain (positive for encapsulated cells), hydrolysis of urea (positive), and final confirmation by using API 20C Aux V3.0 (bioMerieux, Marcy l’Etoile, France). Canavanine-glycine-bromthymol blue agar was used to differentiate between C. gattii and C. neoformans (5). Molecular typing by restriction fragment length polymorphism was used to definitively speciate and subtype C. gattii (6). Fungal culture showed heavy growth of Cryptococcus spp. from the dam (lungs, mediastinal lymph nodes, and placenta) and fetus (mediastinal lymph nodes). Genotyping of primary isolates identified VGIIa C. gattii in both animals. Test results for enteric pathogens, intestinal nematodes, morbillivirus, and Brucella spp. were negative. Fetal infection was most likely hematogenous, disseminated from a primary maternal pulmonary source to the uterus and subsequently to placental vasculature and internal fetal tissues. Infection by aspiration or ingestion of contaminated amniotic fluid was also possible. Although close evaluation of the lung did not show any discernible yeasts, the organism may have been present in an area other than that sectioned. During 1998–2007, ≈450 harbor and Dall’s porpoises (Phocoenoides dalli) and Pacific white-sided dolphins (Lagenorhynchus obliquidens) along the Pacific Northwest Coast were recovered and subjected to necropsy. Disseminated cryptococcosis caused by C. gattii since 2000 was diagnosed in 15 harbor porpoises, 10 Dall’s porpoises, 2 adult Pacific white-sided dolphins, and 3 unrecorded species (10 females, 15 males, and 5 unknown sex; 24 adults, 4 juveniles, 1 fetus, and 1 undocumented age; S. Raverty, unpub. data). Wild porpoises in the Pacific Northwest Region, being near shore inhabitants in waters surrounding Vancouver Island, may come into contact with air containing C. gattii at the air–water interface or ingest seawater containing yeasts while feeding (7). Their proximity to a habitat containing Coastal Douglas fir (Pseudotsuga menziesii) and Western hemlock (Tsuga heterophylla) may play a role in the epidemiology of C. gattii because these trees have been associated with cases of C. gattii (8). Cryptococcal infection during pregnancy has been reported in humans and horses (9,10). This fetal case of cryptococcosis may have major human and animal health implications. Further studies should be undertaken to assess possible fetal involvement, identify infections in pregnant females, and provide information on risk reduction and improving diagnosis and treatment.

Increased harbor porpoise mortality in the Pacific Northwest, USA: understanding when higher levels may be normal

In 2006, a marked increase in harbor porpoise Phocoena phocoena strandings were reported in the Pacific Northwest of the USA, resulting in the declaration of an unusual mortality event (UME) for Washington and Oregon to facilitate investigation into potential causes. The UME was in place during all of 2006 and 2007, and a total of 114 porpoises stranded during this period. Responders examined 95 porpoises; of these, detailed necropsies were conducted on 75 animals. Here we review the findings related to this event and how these compared to the years immediately before and after the UME. Relatively equal numbers among sexes and age classes were represented, and mortalities were attributed to a variety of specific causes, most of which were categorized as trauma or infectious disease. Continued monitoring of strandings during 4 yr following the UME showed no decrease in occurrence. The lack of a single major cause of mortality or evidence of a significant change or event, combined with high levels of strandings over several post-UME years, demonstrated that this was not an actual mortality event but was likely the result of a combination of factors, including: (1) a growing population of harbor porpoises; (2) expansion of harbor porpoises into previously sparsely populated areas in Washingtons inland waters; and (3) a more well established stranding network that resulted in better reporting and response. This finding would not have been possible without the integrated response and investigation undertaken by the stranding network.

Fecal pathogen pollution: sources and patterns in water and sediment samples from the upper Cook Inlet, Alaska ecosystem

Fecal pathogens are transported from a variety of sources in multi-use ecosystems such as upper Cook Inlet (CI), Alaska, which includes the states urban center and is highly utilized by humans and animals. This study used a novel water quality testing approach to evaluate the presence and host sources of potential fecal pathogens in surface waters and sediments from aquatic ecosystems in upper CI. Matched water and sediment samples, along with effluent from a municipal wastewater treatment facility, were screened for Salmonella spp., Vibrio spp., Cryptosporidium spp., Giardia spp., and noroviruses. Additionally, Bacteroidales spp. for microbial source tracking, and the fecal indicator bacteria Enterococcus spp. as well as fecal coliforms were evaluated. Overall, Giardia and Vibrio were the most frequently detected potential pathogens, followed by Cryptosporidium and norovirus, while Salmonella was not detected. Sample month, matrix type, and recent precipitation were found to be significant environmental factors for protozoa or host-associated Bacteroidales marker detection, whereas location and water temperature were not. The relative contribution of host-associated markers to total fecal marker concentration was estimated using a Monte Carlo method, with the greatest relative contribution to the Bacteroidales marker concentration coming from human sources, while the remainder of the universal fecal host source signal was uncharacterized by available host-associated assays, consistent with wildlife fecal sources. These findings show how fecal indicator and pathogen monitoring, along with identifying contributing host sources, can provide evidence of coastal pathogen pollution and guidance as to whether to target human and/or animal sources for management.

Seasonal and developmental differences in blubber stores of beluga whales in Bristol Bay, Alaska using high-resolution ultrasound

Diving mammals use blubber for a variety of structural and physiological functions, including buoyancy, streamlining, thermoregulation, and energy storage. Estimating blubber stores provides proxies for body condition, nutritional status, and health. Blubber stores may vary topographically within individuals, across seasons, and with age, sex, and reproductive status; therefore, a single full-depth blubber biopsy does not provide an accurate measure of blubber depth, and additional biopsies are limited because they result in open wounds. We examined high-resolution ultrasound as a noninvasive method for assessing blubber stores by sampling blubber depth at 11 locations on beluga whales in Alaska. Blubber mass was estimated as a proportion of body mass (40% from the literature) and compared to a function of volume calculated using ultrasound blubber depth measurements in a truncated cone. Blubber volume was converted to total and mass-specific blubber mass estimates based on the density of beluga blubber. There was no significant difference in mean total blubber mass between the 2 estimates (R2 = 0.88); however, body mass alone predicted only 68% of the variation in mass-specific blubber stores in juveniles, 7% for adults in the fall, and 33% for adults in the spring. Mass-specific blubber stores calculated from ultrasound measurements were highly variable. Adults had significantly greater blubber stores in the fall (0.48 ± 0.02 kg/kgMB) than in the spring (0.33 ± 0.02 kg/kgMB). There was no seasonal effect in juveniles. High-resolution ultrasound is a more powerful, noninvasive method for assessing blubber stores in wild belugas, allowing for precise measurements at multiple locations.

Application of real-time quantitative PCR assays for detecting marine Brucella spp. in fish:

Brucella ceti and Brucella pinnipedialis have been documented as occurring in marine mammals, and B. ceti has been identified in 3 naturally acquired human cases. Seroconversion and infection patterns in Pacific Northwest harbor seals (Phoca vitulina richardii) and North Atlantic hooded seals (Cystophora cristata) indicate post-weaning exposure through prey consumption or lungworm infection, suggesting fish and possibly invertebrates play an epizootiologic role in marine Brucella transmission and possible foodborne risk to humans. We determined if real-time quantitative PCR (qPCR) assays can detect marine Brucella DNA in fish DNA. Insertion sequence (IS)711 gene and sequence type (ST)27 primer–probe sets were used to detect Brucella associated with marine mammals and human zoonotic infections, respectively. First, DNA extracts from paired-species fish (containing 2 species) samples were tested and determined to be Brucella DNA negative using both IS711 and ST27 primer–probe sets. A representative paired-species fish DNA sample was spiked with decreasing concentrations of B. pinnipedialis DNA to verify Brucella detection by the IS711 primer–probe within fish DNA. A standard curve, developed using isolated DNA from B. pinnipedialis, determined the limit of detection. Finally, the IS711 primer–probe was used to test Atlantic cod (Gadus morhua) DNA extracts experimentally infected with the B. pinnipedialis hooded seal strain. In culture-positive cod tissue, the IS711 limit of detection was ~1 genome copy of Brucella. Agreement between culture and PCR results for the 9 positive and 9 negative cod tissues was 100%. Although a larger sample set is required for validation, our study shows that qPCR can detect marine Brucella in fish.

VARIATION IN HEMATOLOGIC AND SERUM BIOCHEMICAL VALUES OF BELUGAS (DELPHINAPTERUS LEUCAS) UNDER MANAGED CARE

Abstract:  Blood analytes are critical for evaluating the general health of cetacean populations, so it is important to understand the intrinsic variability of hematology and serum chemistry values. Previous studies have reported data for follow-up periods of several years in managed and wild populations, but studies over long periods of time (>20 yr) have not been reported. The study objective was to identify the influences of partitioning characteristics on hematology and serum chemistry analytes of apparently healthy managed beluga (Delphinapterus leucas). Blood values from 31 managed belugas, at three facilities, collected over 22 yr, were assessed for seasonal variation and aging trends, and evaluated for biologic variation among and within individuals. Linear mixed effects models assessed the relationship between the analytes and sex, age, season, facility location, ambient air temperature, and photoperiod. Sex differences in analytes and associations with increasing age were observed. Seasonal variation was observed for hemoglobin, hematocrit, mean corpuscular volume, monocytes, alkaline phosphatase, total bilirubin, cholesterol, and triglycerides. Facilities were associated with larger effects on analyte values compared to other covariates, whereas age, sex, and ambient temperature had smaller effects compared to facility and season. Present findings provide important baseline information for future health monitoring efforts. Interpretation of blood analytes and animal health in managed and wild populations over time is aided by having available typical levels for the species and reference intervals for the degree to which individual animals vary from the species average and from their own baseline levels during long-term monitoring.

VAGINAL CALCULI IN A JUVENILE HARBOR PORPOISE (PHOCOENA PHOCOENA)

Abstract:  A large number of vaginal calculi were observed in a juvenile harbor porpoise (Phocoena phocoena) stranded on Whidbey Island, Washington. Vaginal calculi have been reported in other species, but not in harbor porpoises. Histologic examination of the urinary tract revealed mucosal hyperplasia most likely attributable to the calculi. The calculi were numerous (>30), composed completely of struvite (magnesium ammonium phosphate), and on culture yielded Enterococcus spp., a bacterium not usually associated with struvite urolith formation in domestic animals. The only other lesion of note was severe hepatic lipidosis, and its relationship to the development of the vaginal calculi is unknown.

Beluga whale, Delphinapterus leucas, satellite-tagging and health assessments in Cook Inlet, Alaska, 1999 to 2002

Cook Inlet beluga whales, Delphinapterus leucas, are currently listed as ‘Endangered’ under the U.S. Endangered Species Act (ESA). The National Marine Fisheries Service (NMFS) began monitoring this population during the 1990s after it was added to the ESA Candidate Species list in 1988. Monitoring efforts included aerial surveys, and in 1995, the first attempts to capture and satellite-tag whales. Working with Canadian scientists and Alaska Native subsistence hunters in 1995 and 1997, tagging methods were adapted to conditions in Cook Inlet (muddy water, extreme tides, and extensive mudflats), culminating in successful capture and tracking of a whale during the summer of 1999. This was followed by three more years of capture and tagging studies during late summer. Tags were attached to 18 whales between 1999 and 2002. We do not have detailed accounts of these later tagging seasons (e.g., similar to the Appendix chronicling events from the 1997 and 1999 seasons in Ferrero et al. (2000)). Litzky et al. (2001) summarized field operations for the 2000 tagging season, but no reports exist for 2001 and 2002. A reanalysis of the tag dataset (Goetz et al. 2012) led to questions about the captures and how tags were programmed during this time period. Given the Cook Inlet population has continued to decline (Hobbs et al. 2015, Shelden et al. 2017), and was listed as an Endangered Distinct Population Segment under the ESA in October 2008 (NOAA 2008), future recommendations for tagging will depend on lessons learned from these past projects. Lacking detailed field reports, we consolidated information from multiple sources. Herein, we bring these varied sources together to provide a thorough documentation of the tagging operations undertaken in Cook Inlet each summer in 2000, 2001, and 2002. We include revised tag transmission timelines, monthly movement maps, dive behavior data, and ice-association graphs and maps for all whales (where applicable) tagged in 1999, 2000, 2001, and 2002. Whale locations were compared to sighting records (opportunistic and systematic) to determine how many whales were likely proximate to tagged whales. Animations of whale movements are available at http://www.afsc.noaa.gov/News/Cook_Inlet_Beluga_Range_Contracted.htm (accessed 17 Aug. 2016).

Multicentric plasmacytoma in a harbor porpoise Phocoena phocoena off the coast of Whidbey Island, Washington State, USA

Necropsy of a female adult pregnant harbor porpoise Phocoena phocoena revealed a multicentric plasmacytoma. The plasmacytoma infiltrated the cranial lung lobes, mediastinal lymph nodes and the spleen. Diagnosis was based on gross, histopathologic and immunohistochemical studies. Histopathology revealed a diffuse proliferation of atypical pleomorphic neoplastic round cells with plasmacytic features. Positive immunohistochemistry with anti-CD79a and anti-CD20 antibody markers and anti-multiple myeloma oncogene 1 (MUM-1) for plasmacytoma confirmed this neoplasm to be of B-cell origin. This is the first recorded case of a plasmacytoma in a harbor porpoise. Routine viral screening was negative via standard PCR for herpesvirus and reverse transcriptase PCR for morbillivirus. Retroviral screening was not performed.