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

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 Washington’s 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.Keywords: Unusual mortality event, Pacific Northwest, Phocoena phocoena, StrandingKeywords: Unusual mortality event, Pacific Northwest, Phocoena phocoena, Strandin

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

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

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

Detection of Pathogenic Leptospira Bacteria in Pinniped Populations via PCR and Identification of a Source of Transmission for Zoonotic Leptospirosis in the Marine Environment▿

Leptospirosis, caused by the spirochete Leptospira, is a geographically widespread disease that affects a broad range of mammals, including marine mammals. Among pinniped populations, periodic epizootics of leptospirosis are responsible for significant die-offs. Along the west coast of North America, the most recent leptospirosis epizootic occurred in 2004, during which samples were collected from cases ranging from California to British Columbia. The primary objective of this study was to use this well-defined sample set to determine the feasibility of using PCR techniques to diagnose Leptospira infection among pinniped populations in comparison with diagnostic methodologies commonly used for marine mammals. Successful amplification was achieved from a variety of samples, including freshly collected urine, urine stored at −80°C for less than 6 months, and kidney (freshly collected, frozen, and decomposed), as well as feces- and urine-contaminated sand collected in the vicinity of a live-stranded animal. Pathological examination of tissue collected from Leptospira-infected animals revealed the presence of leptospiral antigen in the kidneys. The use of species-specific primer pairs revealed a pattern of host specificity for Leptospira interrogans in sea lions and Leptospira kirschneri in elephant seals. These studies indicate PCR is a sensitive and specific diagnostic tool for the detection of Leptospira infection in pinnipeds and reveal a potential source for epizootic, enzootic, and zoonotic spread of leptospirosis in a marine environment

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