Preliminary Investigation into the Microbial Culture and Molecular Screening of Exhaled Breaths of Southern Resident Killer Whales (Orcinus Sp) and Pathogen Screening of the Sea-Surface Microlayer (SML) and Sub-Surface Water Samples in Washington State
IAAAM Archive
S. Raverty1; E. Zabek1; J.P. Schroeder2; R. Wood3; D.E. Bain4; C.E. Cameron5
1Animal Health Center, British Columbia Ministry of Agriculture, Food and Fisheries, Abbotsford, BC, Canada; 2Marine Mammal Research Associates, Sequim WA, USA and Global Research and Rescue, Seattle, WA, USA; 3Global Research and Rescue, Seattle, WA, USA; 4Friday Harbor Laboratories, University of Washington, Friday Harbor, WA, USA and Global Research and Rescue, Inc., Seattle, WA, USA; 5Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada


Within the Northeastern Pacific, there are three killer whale (Orcinus) ecotypes; the transients, southern residents/northern residents and offshores. Between 1995 and 2001, there was precipitous decline in the population of southern resident killer whales from 98 to 79 individuals. To place this decline in a global perspective, necropsy reports, published articles, and stranding records were reviewed and post mortem findings documented. Of 222 documented killer whale strandings between 1944 and 2003, histopathology and bacteriology were conducted on 46 (23%) of the animals. Subacute to chronic pneumonia was the most common morphologic diagnosis in these animals (23/46, 50%). In 14/23 (61%) of these whales, the inflammation would have been sufficiently severe to account for the proximate cause of death. Primary pathogens included Aspergillus spp., Candida spp, Erysipelothrix rhusiopathiae, Pseudomonas spp, Staphylococcus aureus, zygomycetes and nonspecific polymicrobial infections.

In order to better characterize potential pathogen burden, exposure and recruitment in resident killer whales, two preliminary field efforts were undertaken to collect exhaled blowhole air samples, as well as air-water interface (sea surface microlayer, SML) and sub-surface water samples for microbial analysis. Exhaled air samples were collected by positioning 4 bacteriology plates secured to an 8 m long aluminum pole and passing them through the exhaled plume over the blowhole of a surfacing whale. The plates (modified to be covered until collection) included: a sterile plate with no media, a blood agar plate, a salt enriched tryptone soya agar (TSA) plate, and a Sabouraud (SAB) media plate.

Once the exhaled air was collected, each sterile plate was aseptically swabbed and samples were inoculated on site into Salmonella enrichment media, fungal media and collected for polymerase chain reaction (PCR). For environmental samples, SML and 0.5 meter deep water was inoculated into TSA supplemented with 2% NaCl, TSA plain, Columbia Blood agar, selenite broth, and SAB agar to quantify total and fecal coliforms and screen for Salmonella spp, Pseudomonas aeruginosa and Clostridium spp. PCR was employed to screen for dolphin and phocid morbillivirus, canine distemper virus, calicivirus, papillomavirus, marine mammal specific Brucella spp, Mycoplasma (Mollicutes), and universal herpesvirus. Swabs were then inoculated into Mabin Dawby and VERO cells and incubated for 3 weeks to assess for cytopathic effect. A set of plates exposed only to ambient air was processed as a control.

Organisms recovered from 6 exhaled breaths include individual isolates of Penicillium sp, Aureobasidium pullulans, Aureobasidium pullulans, Alternaria sp, Trametes versicolour, Penicillium brevicompactum, Cladosprium cladosporiodes, Pseudomonas fluorescens, Pencillium glabrum, Staphylococcus epidermidis, S pasteuri, S xylosus, Bacillacae bacterium, Staphylococcus sp and Rothia dentocariosa. Isolates were initially identified biochemically, then by sequencing of 16s DNA. No growth was recovered in 3 animals, 1 week post sampling.

Environmental growth included Vibrio tasmaniensis, Vibrio logei, Pseudoalteromonas sp, Photobacterium sp, Vibrio sp., Moritella marina, Bacillacae bacterium, Macrococcus equipericicus, Bacillus simplex, Macrococcus caseolyticus, Hypocrea sp, Penicillium purpurogenum, Alternaria sp., Penicillium namyslowski, Psychrobacter immobilis, Burkholderia glumae, Burkholderia glumae, Vibrio wodanis, Exiguobacterium sp, Halomonas sp., Pseudoalteromonas haloplanktis, Aeromonas sp., Moritella sp and Photobacterium phosphoreum, Photobacter damselae, Burkholderia glumae, Vibrio logei, Pseudoalteromons arctica, Vibrio tasmamaniensis, and Pseudoalteromonas sp. Interestingly, there were 4 environmental isolates of Aspergillus fumigatus, a recognized pathogen of multiple captive and wild marine mammal species. All samples were negative for screened pathogens by PCR and no cytopathic effect was detected in cell culture.

With expanding population growth, industrial development and agricultural intensification, particularly within south and central Puget Sound, Washington State, there are increased environmental stressors and perceived anthropogenic effects on killer whale (and harbor seal) populations which may impact reproductive performance, immune suppression and potentially predispose these animals to infectious disease. Although preliminary, these field investigations provide some baseline information on the microbial and environmental flora of killer whales, recently listed as endangered under the ESA and SARA, and parts of the habitat they transit during each year, Puget Sound and Georgia Basin.

These studies were conducted under NOAA Permit #965-1821-00 and WDFW Permit #06-322.

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Stephen A. Raverty

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