Risk factor determination and qualitative risk assessment of Mucormycosis in Harbor Porpoise, an emergent fungal disease in Salish Sea marine mammals

Mucorales infections are increasing in frequency and are a One Health pathogen of concern. In humans and domestic animals, risk factors include being immunocompromised, elevated circulating serum iron, contaminated open wounds, or metabolic diseases such as ketoacidosis or uncontrolled diabetes. Mucormycosis was first identified in 2012 in Pacific Northwest marine mammals, predominantly in harbor porpoises. We performed an assessment to determine the overall qualitative risk, or risk score, of mucormycosis in harbor porpoises. Risk factors for this disease are unknown in aquatic mammals. In a separate risk factor analysis, potential risk factors such as pollutants, trace metals (e.g., iron), and co-infection with other pathogens (e.g., viruses and Brucella spp.) were examined in mucormycosis cases and noncases using a matched case-control study design, to determine the presence and strength of association of these factors with mucormycosis. Disease severity (gross and histopathology) and exposure scores were multiplied together to obtain the overall risk scores of 9 -16 which corresponded to moderate and severe, respectively. In the risk factor analysis, the factors most strongly associated with a mucormycosis case, relative to a control, were elevated liver iron, decreased blubber thickness, and the decreased ratio of the sum of PCB congeners/sum of PBDE congeners. The results of this study suggest that mucormycosis may pose an inordinately high risk to harbor porpoises (and potentially sympatric species in the Salish Sea such as southern resident killer whales) based on the detected prevalence and the severity of lesions observed at necropsy. However, the risk may be greater on an individual basis compared to the overall population, and is likely related to other factors such as increased POP and heavy metal burdens

Self-Mating in the Definitive Host Potentiates Clonal Outbreaks of the Apicomplexan Parasites Sarcocystis neurona and Toxoplasma gondii

Tissue-encysting coccidia, including Toxoplasma gondii and Sarcocystis neurona, are heterogamous parasites with sexual and asexual life stages in definitive and intermediate hosts, respectively. During its sexual life stage, T. gondii reproduces either by genetic out-crossing or via clonal amplification of a single strain through self-mating. Out-crossing has been experimentally verified as a potent mechanism capable of producing offspring possessing a range of adaptive and virulence potentials. In contrast, selfing and other life history traits, such as asexual expansion of tissue-cysts by oral transmission among intermediate hosts, have been proposed to explain the genetic basis for the clonal population structure of T. gondii. In this study, we investigated the contributing roles self-mating and sexual recombination play in nature to maintain clonal population structures and produce or expand parasite clones capable of causing disease epidemics for two tissue encysting parasites. We applied high-resolution genotyping against strains isolated from a T. gondii waterborne outbreak that caused symptomatic disease in 155 immune-competent people in Brazil and a S. neurona outbreak that resulted in a mass mortality event in Southern sea otters. In both cases, a single, genetically distinct clone was found infecting outbreak-exposed individuals. Furthermore, the T. gondii outbreak clone was one of several apparently recombinant progeny recovered from the local environment. Since oocysts or sporocysts were the infectious form implicated in each outbreak, the expansion of the epidemic clone can be explained by self-mating. The results also show that out-crossing preceded selfing to produce the virulent T. gondii clone. For the tissue encysting coccidia, self-mating exists as a key adaptation potentiating the epidemic expansion and transmission of newly emerged parasite clones that can profoundly shape parasite population genetic structures or cause devastating disease outbreaks

PCBs and PBDEs in Salish Sea harbour seals from 1984 to 2014: notable improvements following regulatory changes

As high trophic level, non-migratory marine mammals, harbour seals (Phoca vitulina) inhabiting the Salish Sea provide an integrated signal of localized food web contamination. Blubber biopsies from harbour seal pups were collected at four sites in the Salish Sea (Hornby Island and Burrard Inlet in Canada; Smith Island and Gertrude Island in the USA) and analyzed for polychlorinated biphenyls (PCBs) and polybrominated diphenylethers (PBDEs). Seals from Gertrude Island had the highest PCB and PBDE concentrations, although spatial variation was less pronounced for PBDEs. PCBs in seals declined between 1984 and 2014, reflecting their regulation in the mid-1970s. PBDEs, however, doubled every 3.1 years between 1984 and 2003, before dropping in 2009. This recent drop may reflect a response to regulatory measures taken in North America after 2004. The application of toxicity reference values (TRVs) for the dominant PCBs to our results reveal an improving “health index” over time in Gertrude Island seals in this study. However, the application of a harbour seal-specific PCB TRV of 1.3 mg/kg revealed that a majority of Gertrude Island seal pups exceeded that threshold in 2003, while seals from the other sites had relatively good health indices. Analyses of 2014 samples are underway and will increase our assessment of temporal trends as well as the effectiveness of PBDE regulations. Our study suggests that regulations have been very effective in reducing health risks to Salish Sea harbour seals, although the slow nature of these improvements underscore the lingering risks associated with very persistent contaminants

Polyparasitism is associated with increased disease severity in Toxoplasma gondii-infected marine sentinel species.

In 1995, one of the largest outbreaks of human toxoplasmosis occurred in the Pacific Northwest region of North America. Genetic typing identified a novel Toxoplasma gondii strain linked to the outbreak, in which a wide spectrum of human disease was observed. For this globally-distributed, water-borne zoonosis, strain type is one variable influencing disease, but the inability of strain type to consistently explain variations in disease severity suggests that parasite genotype alone does not determine the outcome of infection. We investigated polyparasitism (infection with multiple parasite species) as a modulator of disease severity by examining the association of concomitant infection of T. gondii and the related parasite Sarcocystis neurona with protozoal disease in wild marine mammals from the Pacific Northwest. These hosts ostensibly serve as sentinels for the detection of terrestrial parasites implicated in water-borne epidemics of humans and wildlife in this endemic region. Marine mammals (151 stranded and 10 healthy individuals) sampled over 6 years were assessed for protozoal infection using multi-locus PCR-DNA sequencing directly from host tissues. Genetic analyses uncovered a high prevalence and diversity of protozoa, with 147/161 (91%) of our sampled population infected. From 2004 to 2009, the relative frequency of S. neurona infections increased dramatically, surpassing that of T. gondii. The majority of T. gondii infections were by genotypes bearing Type I lineage alleles, though strain genotype was not associated with disease severity. Significantly, polyparasitism with S. neurona and T. gondii was common (42%) and was associated with higher mortality and more severe protozoal encephalitis. Our finding of widespread polyparasitism among marine mammals indicates pervasive contamination of waterways by zoonotic agents. Furthermore, the significant association of concomitant infection with mortality and protozoal encephalitis identifies polyparasitism as an important factor contributing to disease severity in marine mammals

Sarcocystis neurona Transmission from Opossums to Marine Mammals in the Pacific Northwest.

Increasing reports of marine mammal deaths have been attributed to the parasite Sarcocystis neurona. Infected opossums, the only known definitive hosts, shed S. neurona sporocysts in their feces. Sporocysts can contaminate the marine environment via overland runoff, and subsequent ingestion by marine mammals can lead to fatal encephalitis. Our aim was to determine the prevalence of S. neurona in opossums from coastal areas of Washington State (USA) and to compare genetic markers between S. neurona in opossums and marine mammals. Thirty-two road-kill opossums and tissue samples from 30 stranded marine mammals meeting inclusion criteria were included in analyses. Three opossums (9.4%) and twelve marine mammals (40%) were confirmed positive for S. neurona via DNA amplification at the ITS1 locus. Genetic identity at microsatellites (sn3, sn7, sn9) and the snSAG3 gene of S. neurona was demonstrated among one harbor porpoise and two opossums. Watershed mapping further demonstrated plausible sporocyst transport pathways from one of these opossums to the location where an infected harbor porpoise carcass was recovered. Our results provide the first reported link between S. neurona genotypes on land and sea in the Pacific Northwest, and further demonstrate how terrestrial pathogen pollution can impact the health of marine wildlife

Sarcocystis neurona Transmission from Opossums to Marine Mammals in the Pacific Northwest.

Increasing reports of marine mammal deaths have been attributed to the parasite Sarcocystis neurona. Infected opossums, the only known definitive hosts, shed S. neurona sporocysts in their feces. Sporocysts can contaminate the marine environment via overland runoff, and subsequent ingestion by marine mammals can lead to fatal encephalitis. Our aim was to determine the prevalence of S. neurona in opossums from coastal areas of Washington State (USA) and to compare genetic markers between S. neurona in opossums and marine mammals. Thirty-two road-kill opossums and tissue samples from 30 stranded marine mammals meeting inclusion criteria were included in analyses. Three opossums (9.4%) and twelve marine mammals (40%) were confirmed positive for S. neurona via DNA amplification at the ITS1 locus. Genetic identity at microsatellites (sn3, sn7, sn9) and the snSAG3 gene of S. neurona was demonstrated among one harbor porpoise and two opossums. Watershed mapping further demonstrated plausible sporocyst transport pathways from one of these opossums to the location where an infected harbor porpoise carcass was recovered. Our results provide the first reported link between S. neurona genotypes on land and sea in the Pacific Northwest, and further demonstrate how terrestrial pathogen pollution can impact the health of marine wildlife

PBDE flame retardants and PCBs in migrating Steller sea lions (Eumetopias jubatus) in the Strait of Georgia, British Columbia, Canada

a b s t r a c t Polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) were measured in blubber biopsy samples from 22 live-captured Steller sea lions (Eumetopias jubatus) that had just entered the Strait of Georgia, British Columbia, Canada, for their overwintering feeding season. P PBDE ranged from 50 lg kg À1 (lipid weight) in adult females to 3780 lg kg À1 in subadult individuals. P PCBs ranged from 272 lg kg À1 in adult females to 14 280 lg kg À1 in subadult individuals. While most PBDE and PCB congeners were transferred through milk to pups, PCBs with log K OW > 7.0 (PCBs 206, 207, 208 and 209) appeared constrained, resulting in a lighter mixture in pups compared to adult females. The ratio of individual PCB congeners by metabolic group (Groups I, II, III, IV and V) to PCB-153 regressed against length of males suggested poor biotransformation of these compounds (slopes did not differ from zero, p > 0.05). PBDE congeners 49, 99, 153 and 183 appeared bioaccumulative (slopes of ratio BDE/PCB 153 versus length were higher than zero, p < 0.05), but the dominance of the single congener, BDE-47 (64% of total PBDEs), likely due in part to debromination pathways, reduced our ability to explore congenerspecific dynamics of PBDEs in these pinnipeds. With 80% of our Steller sea lions exceeding a recent toxicity reference value for PCBs, the fasting-associated mobilization of these contaminants raises concerns about a heightened vulnerability to adverse effects during annual migrations

Reemergence of Guadalupe Fur Seals in the U.S. Pacific Northwest: The Epidemiology of tranding Events During 2005–2016

Guadalupe fur seals (GFS), Arctocephalus philippii townsendi, an U.S. Endangered Species Act threatened pinniped, have recently reappeared in their historic range along the western seaboard of the continental United States. Starting 2005 through 2016, 169 GFSs stranded in Washington and Oregon, involving two designated unusual mortality events. The circumstances surrounding GFS strandings, mortality, and their increased presence in Oregon and Washington were analyzed during this study. Detailed necropsies, histopathology (n = 93), and epidemiological analysis found three main causes of death (COD): emaciation (44%), trauma (29%), and infectious disease (19%) and the factors associated with overall strandings and emaciation. Trauma included many cases found associated with fisheries interactions and clustered near the mouth of the Columbia River, where high levels of commercial fishing occur. The most common pathogens found associated with disease were Toxoplasma gondii, Sarcocystis neurona, and gastrointestinal helminths. Seasonality and upwelling were associated with higher stranding numbers regardless of COD. Seasonal migration into the region, coinciding with postweaning, suggests young GFSs are in search of prey and habitat resources. Reemergence of GFSs is likely due to conservation efforts, which have been critical for species recovery in the region. Continued monitoring is needed as this vulnerable species continues to rebound