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Purpose: Raf-kinases include three major isoforms. Although the role of B-Raf in melanoma is well established, little is known about C-Raf. We studied effects of C-Raf knockdown in vitro and assessed expression of C-Raf in a large cohort of melanomas and nevi.Experimental Design: Using specific siRNAs, we knocked down C-Raf expression, and determined the effect on viability, MAP extracellular signal-regulated kinase (ERK)/ERK kinase signaling, and apoptosis in seven melanoma cell lines. We determined the IC50 of the C-Raf inhibitors sorafenib and GW5074, and studied the effects of GW5074 on cell signaling. Using an automated method to measure in situ protein expression, we quantified C-Raf expression in 263 nevi and 523 melanomas.Results: C-Raf was knocked down in three cell lines with detectable phospho-C-Raf, resulting in decreased viability in two of the three (YULAC and YUROB). This resulted in decreased Bcl-2 expression and phospho-Bad cleavage, without affecting phospho-MEK and phospho-ERK. Sensitivity to sorafenib and GW5074 varied. GW5074 inhibited mitogen-activated protein kinase signaling without Bcl-2 and phospho-Bad down-regulation. C-Raf was highly expressed in melanomas compared with nevi (P < 0.0001), and no nevi had high C-Raf expression. C-Raf expression was higher in metastatic than primary specimens (P = 0.0225).Conclusions: C-Raf siRNA knock-down results in decreased viability of YULAC (B-RafV600K) and YUROB (B-RafWT) melanoma cells, likely mediated by Bcl-2 inhibition rather than mitogen-activated protein kinase inhibition. Cotargeting C-Raf and parallel pathways might be an effective therapeutic approach for melanoma. C-Raf expression is up-regulated in a subset of melanomas but not in nevi, suggesting that it might be a valuable diagnostic marker and therapeutic target. (Clin Cancer Res 2009;15(18):5704–13)

Supplementary Data from C-Raf Is Associated with Disease Progression and Cell Proliferation in a Subset of Melanomas

How virulent parasites are maintained in nature is an important paradigm of eukaryotic pathogenesis. Here we used population genetics and molecular methods to study the evolution and emergence of genetic variants of the protozoan parasiteToxoplasma gondii,referred collectively as Type X (HG12), recovered from a threatened marine mammal species. Specifically, 53T. gondiistrains were isolated from southern sea otters (SSO) that stranded between 1998-2004 withT. gondiiinfection (ranging from chronic incidental infections to fatal encephalitis). Over 74% of these SSO, collected throughout their geographic range, were infected with Type X, based on multi-locus PCR-DNA sequencing. Depending on the locus investigated, Type X strains possessed one of three allelic types that had independently assorted across the strains examined; either genetically distinct alleles, referred to as “ψ” or “8”, or a Type II allele. Phylogenetic incongruence among locus-specific trees, genome-wide CGH array and WGS analyses confirmed that Type X is a sexual clade of natural recombinants that resemble F1 progeny from a genetic cross between Type II and a mosaic of two distinct “ψ” or “δ” ancestries. A single Type X genotype (19/53; 36%) that largely caused subclinical chronic infections in SSO, was highly pathogenic to mice (LD100= 1 parasite). To determine whether murine virulence genes could be mapped within this population of natural isolates, we performed a genome scan and identified four QTLs with LOD scores greater than 4.0. Targeted disruption of ROP33, the strongest candidate from among 16 genes within the highest QTL on Chromosome VIIa established ROP33 as a murine virulence locus. The ability of this highly pathogenic mouse-virulentT. gondiiclone to expand its environmental niche and infect a majority of SSO supports a virulence shift model whereby generalist pathogens likeT. gondiiutilize their sexual cycles to produce new strains that possess an expanded biological potential. Such a trait enables pathogens to extend their host range or be naturally selected within their vast intermediate host range to maximize transmission. Our work establishes a rationale for how virulent strains can be maintained cryptically in nature across a pathogen’s broad host range, and act as potential reservoirs for epidemic disease.ImportanceWaterborne outbreaks of protozoal parasites are capable of causing fatal disease in a wide range of animals, including humans. Population expansion of felids in addition to anthropogenic changes near marine estuarine environments may facilitate marine wildlife exposure to highly infectiousToxoplasma gondiioocysts shed in felid feces. Infected cats shed millions of environmentally-resistantT. gondiioocysts that can be widely dispersed by storm events. In North AmericaT. gondiiis thought to possess a highly clonal population structure dominated by 4 clonal lineages (I, II, III, and X). Population genetic analysis of 53T. gondiiisolates collected longitudinally from SSO infected withT. gondiithat stranded between 1998-2004 identified 74% of otters infected with Type XT. gondii, and that Type X is not a clonal lineage, but rather a recombinant clade of at least 12 distinct strains consistent with a recent genetic cross. Importantly, one Type X haplotype was isolated from 36% of southern sea otters (Enhydra lutris neries) across their geographic range in California. This haplotype was highly pathogenic to mice but caused relatively benign infections in SSO. A genome scan was performed to identify a virulence locus; a secreted serine threonine kinase (ROP33) that enhanced pathogenicity in laboratory mice, but not sea otters. Our data support a virulence shift model whereby generalist pathogens likeT. gondiiutilize their sexual cycles to produce virulent strains that can be maintained cryptically in nature, according to their differential capacity to cause disease within the pathogen’s broad intermediate host range. This type of “host selection” has important public health implications. Strains capable of causing fatal infections can persist in nature by circulating as chronic infections in resistant intermediate host species that act as reservoirs for epidemic disease.

IntroductionDebate around a common definition of global health has seen extensive scholarly interest within the last two decades; however, consensus around a precise definition remains elusive. The objective of this study was to systematically review definitions of global health in the literature and offer grounded theoretical insights into what might be seen as relevant for establishing a common definition of global health.MethodA systematic review was conducted with qualitative synthesis of findings using peer-reviewed literature from key databases. Publications were identified by the keywords of ‘global health’ and ‘define’ or ‘definition’ or ‘defining’. Coding methods were used for qualitative analysis to identify recurring themes in definitions of global health published between 2009 and 2019.ResultsThe search resulted in 1363 publications, of which 78 were included. Qualitative analysis of the data generated four theoretical categories and associated subthemes delineating key aspects of global health. These included: (1) global health is a multiplex approach to worldwide health improvement taught and pursued at research institutions; (2) global health is an ethically oriented initiative that is guided by justice principles; (3) global health is a mode of governance that yields influence through problem identification, political decision-making, as well as the allocation and exchange of resources across borders and (4) global health is a vague yet versatile concept with multiple meanings, historical antecedents and an emergent future.ConclusionExtant definitions of global health can be categorised thematically to designate areas of importance for stakeholders and to organise future debates on its definition. Future contributions to this debate may consider shifting from questioning the abstract ‘what’ of global health towards more pragmatic and reflexive questions about ‘who’ defines global health and towards what ends.

Island fox (Urocyon littoralis) populations on four California Channel Islands have declined severely since 1994. Canine distemper (CDV) was suspected to be responsible for the decline of the Santa Catalina Island fox, so knowledge of infectious disease exposure in the remaining island fox populations was urgently needed. This study reviewed previous pathogen exposure in island foxes and investigated the current threat by conducting a serologic survey of foxes on all islands and sympatric feral cats on three islands from 2001 to 2003 for antibodies against canid pathogens. Before the decline, foxes had evidence of exposure to CDV, canine adenovirus (CAV), canine parvovirus (CPV), and Toxoplasma, with exposure to these five pathogens differing greatly by island. Exposure to canine coronavirus (CCV), canine herpesvirus (CHV), and Leptospira was rare. In 2001–2003, wild-born foxes had evidence of exposure to CDV (5.2–32.8%) on 5 of 6 islands, CPV (28–100%) and CAV (4.7–100%) on five islands, and Toxoplasma gondii (2.3–15.4%) on four islands. Exposure to CCV, CHV and Leptospira was less common. Sharing of infectious agents between sympatric foxes and feral cats appeared minimal, but CDV exposure was detected in two cats on Santa Catalina Island. Domestic dogs have historically been present on the islands, but it is not known if canine diseases can be maintained in fox populations without the continual presence of dogs. Targeted vaccination programs against the most virulent pathogens and continued intensive disease surveillance may help protect the critically small remaining fox populations from disease outbreaks that could threaten the success of ongoing conservation efforts.

Sarcocystis neurona was recognised as an important cause of mortality in southern sea otters (Enhydra lutris nereis) after an outbreak in April 2004 and has since been detected in many marine mammal species in the Northeast Pacific Ocean. Risk of S. neurona exposure in sea otters is associated with consumption of clams and soft-sediment prey and is temporally associated with runoff events. We examined the spatial distribution of S. neurona exposure risk based on serum antibody testing and assessed risk factors for exposure in animals from California, Washington, British Columbia and Alaska. Significant spatial clustering of seropositive animals was observed in California and Washington, compared with British Columbia and Alaska. Adult males were at greatest risk for exposure to S. neurona, and there were strong associations with terrestrial features (wetlands, cropland, high human housing-unit density). In California, habitats containing soft sediment exhibited greater risk than hard substrate or kelp beds. Consuming a diet rich in clams was also associated with increased exposure risk. These findings suggest a transmission pathway analogous to that described for Toxoplasma gondii, with infectious stages traveling in freshwater runoff and being concentrated in particular locations by marine habitat features, ocean physical processes, and invertebrate bioconcentration.

OBJECTIVE To describe the general seroprevalence of anti-Sarcocystis neurona and anti-Neospora hughesi antibodies among healthy equids by use of indirect fluorescent antibody tests and determine potential risk factors for seropositivity. DESIGN Cross-sectional study. SAMPLE Whole blood samples collected from 5,250 equids (1 sample/animal) across 18 states in the United States during October 2013. PROCEDURES Information regarding potential risk factors (geographic region, breed, primary use, sex, and age) was collected along with the blood samples. For each equid, an indirect fluorescent antibody test was used to determine serum titers of antibody against each of the 2 protozoal parasites. Mixed-effects logistic regression models were created to determine ORs for seropositivity. RESULTS The overall seroprevalence of anti-S neurona and anti-N hughesi antibodies in the tested equids was 78% and 34%, respectively. Of the equids, 31% were seropositive and 18% were seronegative for antibodies against both parasites. Factors associated with equids being seropositive for anti-S neurona antibodies were residence in the South, warmblood breed, and age > 5 years. Seroprevalence of anti-N hughesi antibodies did not differ among equids in different states across the country, but warmblood breed and age > 5 years were associated with seropositivity. CONCLUSIONS AND CLINICAL RELEVANCE With regard to risk factors for S neurona and N hughesi exposure and antibody response among tested equids, older age was not unexpected; however, the influences of warmblood breed and geographic location on seropositivity for anti-S neurona antibody but not for anti-N hughesi antibody deserve further investigation.

The One Health approach has gained support across a range of disciplines; however, training opportunities for professionals seeking to operationalize the interdisciplinary approach are limited. Academic institutions, through the development of high-quality, experiential training programs that focus on the application of professional competencies, can increase accessibility to One Health education. The Rx One Health Summer Institute, jointly led by US and East African partners, provides a model for such a program. In 2017, 21 participants representing five countries completed the Rx One Health program in East Africa. Participants worked collaboratively with communities neighboring wildlife areas to better understand issues impacting human and animal health and welfare, livelihoods, and conservation. One Health topics were explored through community engagement and role-playing exercises, field-based health surveillance activities, laboratories, and discussions with local experts. Educational assessments reflected improvements in participants’ ability to apply the One Health approach to health and disease problem solving, as well as anticipate cross-sectoral challenges to its implementation. The experiential learning method, specifically the opportunity to engage with local communities, proved to be impactful on participants’ cultural awareness. The Rx One Health Summer Institute training model may provide an effective and implementable strategy by which to contribute to the development of a global One Health workforce.

We investigated the prevalence of Toxoplasma gondii in 2011-15 to assess its potential threat on the endangered Amargosa vole ( Microtus californicus scirpensis) in California, US. Surveillance was simultaneously performed on populations of syntopic rodent species. We detected antibodies to T. gondii in sera from 10.5% of 135 wild-caught Amargosa voles; 8% of 95 blood samples were PCR-positive for the T. gondii B1 gene, and 5.0% of 140 sympatric rodent brain samples were PCR-positive. Exposure to T. gondii did not change the probability that an animal would be recaptured in the field study. Behavioral response to domestic cat ( Felis catus ) and bobcat ( Lynx rufus ) urine was evaluated in five nonendangered Owens Valley voles ( Microtus californicus vallicola) as surrogates for Amargosa voles and seven uninfected controls. Voles showed mild attraction to mouse urine and had neutral reactions to domestic cat urine whether or not infected. Time spent near bobcat urine was approximately twice as high in infected than in uninfected voles (although not statistically significant). The presence of T. gondii in wild Amargosa vole and sympatric rodent populations may hinder the endangered Amargosa vole population's ability to recover in the wild.

Cryptosporidium and Giardia spp. are waterborne, fecally-transmitted pathogens that cause economic loss due to gastroenteritis and beach closures. We applied quantitative microbial risk assessment (QMRA) to determine the health risks for humans and sea otters due to waterborne exposure of Cryptosporidium and Giardia spp. when swimming in three types of surface waters: river, stormwater and wastewater effluent during the wet and dry seasons in the central coast of California. This is the first application of QMRA to estimate both the probability of infection in Southern sea otters and the probability of illness in humans, using microbial source tracking (MST) as a variable. Children swimming close to stormwater discharges had an estimated Cryptosporidium-associated illness probability that exceeded the accepted U.S. EPA criteria (32 illnesses/1000 swimmers or 3.2%). Based on the assumption that sea otters are as susceptible as humans to Cryptosporidium infection, the infection probabilities were close to 2% and 16% when sea otters were swimming at the end of points of rivers and stormwater discharges, respectively. In the case of Giardia, infection probabilities of 11% and 23% were estimated for sea otters swimming at the end of point of wastewater discharges, assuming that sea otters are as susceptible as gerbils and humans, respectively. The results of this QMRA suggest that 1) humans and sea otters are at risk when swimming at outflow sites for rivers, stormwater and treated wastewater effluent; 2) reduced loads of viable protozoan cysts and oocysts in recreational water can lessen the probability of infection of humans and sea otters; and 3) the risk of infection of humans and sea otters can be reduced with the treatment of wastewater to decrease oocyst and cyst viability before effluent is released into the sea.

We used a community engagement approach to develop a One Health profile of an agro-pastoralist population at the interface of wildlife areas in eastern South Africa. Representatives from 262 randomly-selected households participated in an in-person, cross-sectional survey. Questions were designed to ascertain the participants' knowledge, attitudes, and practices with regard to human health, domestic animal health, and natural resources including wildlife and water. Surveys were conducted within four selected villages by a team of trained surveyors and translators over four weeks in July-August 2013. Questions were a combination of multiple choice (single answer), multiple selection, open-ended, and Likert scale. The study found that nearly three-quarters of all households surveyed reported owning at least one animal (55% owned chickens, 31% dogs, 25% cattle, 16% goats, 9% cats, and 5% pigs). Among the animal-owning respondents, health concerns identified included dissatisfaction with government-run cattle dip facilities (97%) and frequent morbidity and mortality of chickens that had clinical signs consistent with Newcastle disease (49%). Sixty-one percent of participants believed that diseases of animals could be transmitted to humans. Ninety-six percent of respondents desired greater knowledge about animal diseases. With regard to human health issues, the primary barrier to health care access was related to transportation to/from the community health clinics. Environmental health issues revealed by the survey included disparities by village in drinking water reliability and frequent domiciliary rodent sightings positively associated with increased household size and chicken ownership. Attitudes towards conservation were generally favorable; however, the community demonstrated a strong preference for a dichotomous approach to wildlife management, one that separated wildlife from humans. Due to the location of the community, which neighbors the Great Limpopo Transfrontier Conservation Area, and the livestock-dependent lifestyle of the resource-poor inhabitants, a One Health approach that takes into consideration the interconnectedness of human, animal, and environmental health is necessary. The community profile described in this study provides a foundation for health research and planning initiatives that are driven by community engagement and consider the multitude of factors affecting health at the human-domestic animal-wildlife interface. Furthermore, it allows for the determination and quantification of the linkages between human, animal, and environmental health.

Babesiosis is a potentially fatal tick-borne zoonotic disease caused by a species complex of blood parasites that can infect a variety of vertebrates, particularly dogs, cattle, and humans. In the United States, human babesiosis is caused by two distinct parasites, Babesia microti and Babesia duncani. The enzootic cycle of B. microti, endemic in the northeastern and upper midwestern regions, has been well characterised. In the western United States, however, the natural reservoir host and tick vector have not been identified for B. duncani, greatly impeding efforts to understand and manage this zoonotic disease. Two and a half decades after B. duncani was first described in a human patient in Washington State, USA, we provide evidence that the enzootic tick vector is the winter tick, Dermacentor albipictus, and the reservoir host is likely the mule deer, Odocoileus hemionus. The broad, overlapping ranges of these two species covers a large portion of far-western North America, and is consistent with confirmed cases of B. duncani in the far-western United States.

Pathogens entering the marine environment as pollutants exhibit a spatial signature driven by their transport mechanisms. The sea otter ( Enhydra lutris ), a marine animal which lives much of its life within sight of land, presents a unique opportunity to understand land–sea pathogen transmission. Using a dataset on Toxoplasma gondii prevalence across sea otter range from Alaska to California, we found that the dominant drivers of infection risk vary depending upon the spatial scale of analysis. At the population level, regions with high T. gondii prevalence had higher human population density and a greater proportion of human-dominated land uses, suggesting a strong role for population density of the felid definitive host of this parasite. This relationship persisted when a subset of data were analysed at the individual level: large-scale patterns in sea otter T. gondii infection prevalence were largely explained by individual exposure to areas of high human housing unit density, and other landscape features associated with anthropogenic land use, such as impervious surfaces and cropping land. These results contrast with the small-scale, within-region analysis, in which age, sex and prey choice accounted for most of the variation in infection risk, and terrestrial environmental features provided little variation to help in explaining observed patterns. These results underscore the importance of spatial scale in study design when quantifying both individual-level risk factors and landscape-scale variation in infection risk.

The coccidian parasite Toxoplasma gondii infects humans and warm-blooded animals worldwide. The ecology of this parasite in marine systems is poorly understood, although many marine mammals are infected and susceptible to clinical toxoplasmosis. We summarized the lesions associated with T. gondii infection in the California sea lion (Zalophus californianus) population and investigated the prevalence of and risk factors associated with T. gondii exposure, as indicated by antibody. Five confirmed and four suspected cases of T. gondii infection were identified by analysis of 1,152 medical records of necropsied sea lions from 1975-2009. One suspected and two confirmed cases were identified in aborted fetuses from a sea lion rookery. Toxoplasmosis was the primary cause of death in five cases, including the two fetuses. Gross and histopathologic findings in T. gondii-infected sea lions were similar to those reported in other marine mammals. The most common lesions were encephalitis, meningitis, and myocarditis. The antibody prevalence in stranded, free-ranging sea lions for 1998-2009 was 2.5% (±0.03%; IgG titer 640). There was an increase in odds of exposure in sea lions with increasing age, suggesting cumulative risk of exposure and persistent antibody over time. The occurrence of disseminated T. gondii infection in aborted fetuses confirms vertical transmission in sea lions, and the increasing odds of exposure with age is consistent with additional opportunities for horizontal transmission in free-ranging sea lions over time. These data suggest that T. gondii may have two modes of transmission in the sea lion population. Overall, clinical disease was uncommon in our study which, along with low prevalence of T. gondii antibody, suggests substantially less-frequent exposure and lower susceptibility to clinical disease in California sea lions as compared to sympatric southern sea otters (Enhydra lutris nereis).

Pathogens entering the marine environment as pollutants exhibit a spatial signature driven by their transport mechanisms. The sea otter (Enhydra lutris), a marine animal which lives much of its life within sight of land, presents a unique opportunity to understand land–sea pathogen transmission. Using a dataset on Toxoplasma gondii prevalence across sea otter range from Alaska to California, we found that the dominant drivers of infection risk vary depending upon the spatial scale of analysis. At the population level, regions with high T. gondii prevalence had higher human population density and a greater proportion of human-dominated land uses, suggesting a strong role for population density of the felid definitive host of this parasite. This relationship persisted when a subset of data were analysed at the individual level: large-scale patterns in sea otter T. gondii infection prevalence were largely explained by individual exposure to areas of high human housing unit density, and other landscape features associated with anthropogenic land use, such as impervious surfaces and cropping land. These results contrast with the small-scale, within-region analysis, in which age, sex and prey choice accounted for most of the variation in infection risk, and terrestrial environmental features provided little variation to help in explaining observed patterns. These results underscore the importance of spatial scale in study design when quantifying both individual-level risk factors and landscape-scale variation in infection risk.

The interface between humans, domestic animals, and wildlife has been implicated in the emergence of infectious diseases and the persistence of endemic human and animal diseases. For individuals who reside at this interface, particularly those in low-resource settings, the development of disease risk assessment and mitigation skills must be prioritized. Using a community engagement-One Health approach, we implemented a training program aimed at advancing these skills among agro-pastoralists living adjacent to conservation areas in South Africa. The program included professional development of local facilitators who then conducted workshops with community members. Workshops used a series of experiential, inquiry-based activities to teach participants the concepts of pathogen transmission and disease risk assessment and mitigation. The program was implemented over four weeks with 10 facilitators and 78 workshop participants. We conducted a within-subjects experimental study using a mixed methods design to evaluate the program in terms of facilitator and participant One Health knowledge and practices. Quantitative data included pre/post written assessments; qualitative data included focus group discussions, semi-structured interviews, and pre/post photographs. Mean post-test scores of facilitators increased by 17% (p=0.0078). For workshop participants, improvements in knowledge were more likely for females than males (OR=7.315, 95% CI=2.258–23.705, p=0.0009) and participants with a higher versus lower education level, albeit borderline non-significant (OR=4.781, 95% CI=0.942–24.264, p=0.0590). Qualitative analysis revealed the implementation of risk mitigation strategies by 98% (60/61) of workshop participants during the three-month follow-up and included improved personal and domestic hygiene practices and enhanced animal housing. Although further evaluation is recommended, this program may be appropriate for consideration as a scalable approach by which to mitigate human and animal infectious disease risk in high-risk/low-resource communities. Keywords: Community engagement, Health promotion, Experiential learning, Risk assessment, Infectious disease, Program evaluation

We characterize Brucella infection in a wild southern sea otter ( Enhydra lutris nereis) with osteolytic lesions similar to those reported in other marine mammals and humans. This otter stranded twice along the central California coast, US over a 1-yr period and was handled extensively at two wildlife rehabilitation facilities, undergoing multiple surgeries and months of postsurgical care. Ultimately the otter was euthanized due to severe, progressive neurologic disease. Necropsy and postmortem radiographs revealed chronic, severe osteoarthritis spanning the proximal interphalangeal joint of the left hind fifth digit. Numerous coccobacilli within the joint were strongly positive on Brucella immunohistochemical labelling, and Brucella sp. was isolated in pure culture from this lesion. Sparse Brucella-immunopositive bacteria were also observed in the cytoplasm of a pulmonary vascular monocyte, and multifocal granulomas were observed in the spinal cord and liver on histopathology. Findings from biochemical characterization, 16S ribosomal DNA, and bp26 gene sequencing of the bacterial isolate were identical to those from marine-origin brucellae isolated from cetaceans and phocids. Although omp2a gene sequencing revealed 100% homology with marine Brucella spp. infecting pinnipeds, whales, and humans, omp2b gene sequences were identical only to pinniped-origin isolates. Multilocus sequence typing classified the sea otter isolate as ST26, a sequence type previously associated only with cetaceans. Our data suggest that the sea otter Brucella strain represents a novel marine lineage that is distinct from both Brucella pinnipedialis and Brucella ceti. Prior reports document the zoonotic potential of the marine brucellae. Isolation of Brucella sp. from a stranded sea otter highlights the importance of wearing personal protective equipment when handling sea otters and other marine mammals as part of wildlife conservation and rehabilitation efforts.

The infection status of harbor seals Phoca vitulina in central California, USA, was evaluated through broad surveillance for pathogens in stranded and wild-caught animals from 2001 to 2008, with most samples collected in 2007 and 2008. Stranded animals from Mendocino County to San Luis Obispo County were sampled at a rehabilitation facility: The Marine Mammal Center (TMMC, n = 175); wild-caught animals were sampled at 2 locations: San Francisco Bay (SF, n = 78) and Tomales Bay (TB, n = 97), that differed in degree of urbanization. Low prevalences of Salmonella, Campylobacter, Giardia, and Cryptosporidium were detected in the feces of stranded and wild-caught seals. Clostridium perfringens and Escherichia coli were more prevalent in the feces of stranded (58% [78 out of 135] and 76% [102 out of 135]) than wild-caught (42% [45 out of 106] and 66% [68 out of 106]) seals, whereas Vibrio spp. were 16 times more likely to be cultured from the feces of seals from SF than TB or TMMC (p < 0.005). Brucella DNA was detected in 3.4% of dead stranded harbor seals (2 out of 58). Type A influenza was isolated from feces of 1 out of 96 wild-caught seals. Exposure to Toxoplasma gondii, Sarcocystis neurona, and type A influenza was only detected in the wild-caught harbor seals (post-weaning age classes), whereas antibody titers to Leptospira spp. were detected in stranded and wild-caught seals. No stranded (n = 109) or wild-caught (n = 217) harbor seals had antibodies to phocine distemper virus, although a single low titer to canine distemper virus was detected. These results highlight the role of harbor seals as sentinel species for zoonotic and terrestrial pathogens in the marine environment.

Why some Toxoplasma gondii -infected southern sea otters ( Enhydra lutris nereis ) develop fatal toxoplasmosis while others have incidental or mild chronic infections has long puzzled the scientific community. We assessed robust datasets on T. gondii molecular characterization in relation to detailed necropsy and histopathology results to evaluate whether parasite genotype influences pathological outcomes in sea otters that stranded along the central California coast. Genotypes isolated from sea otters were also compared with T. gondii strains circulating in felids from nearby coastal regions to assess land-to-sea parasite transmission. The predominant T. gondii genotypes isolated from 135 necropsied sea otters were atypical Type X and Type X variants (79%), with the remainder (21%) belonging to Type II or Type II/X recombinants. All sea otters that died due to T. gondii as a primary cause of death were infected with Type X or X-variant T. gondii strains. The same atypical T. gondii strains were detected in sea otters with fatal toxoplasmosis and terrestrial felids from watersheds bordering the sea otter range. Our results confirm a land–sea connection for virulent T. gondii genotypes and highlight how faecal contamination can deliver lethal pathogens to coastal waters, leading to detrimental impacts on marine wildlife.

Mountain gorillas (Gorilla beringei beringei) are critically endangered primates surviving in two isolated populations in protected areas within the Virunga Massif of Rwanda, Uganda, the Democratic Republic of Congo, and in Bwindi Impenetrable National Park in Uganda. Mountain gorillas face intense ecologic pressures due to their proximity to humans. Human communities outside the national parks, and numerous human activities within the national parks (including research, tourism, illegal hunting, and anti-poaching patrols), lead to a high degree of contact between mountain gorillas and wildlife, domestic animals, and humans. To assess the pathogen transmission potential between wildlife and livestock, feces of mountain gorillas, forest buffalo (Syncerus caffer nanus), and domestic cattle (Bos taurus) in Rwanda were examined for the parasites Giardia and Cryptosporidium. Giardia was found in 9% of mountain gorillas, 6% of cattle, and 2% of forest buffalo. Our study represents the first report of Giardia prevalence in forest buffalo. Cryptosporidium-like particles were also observed in all three species. Molecular characterization of Giardia isolates identified zoonotic genotype assemblage B in the gorilla samples and assemblage E in the cattle samples. Significant spatial clustering of Giardia-positive samples was observed in one sector of the park. Although we did not find evidence for transmission of protozoa from forest buffalo to mountain gorillas, the genotypes of Giardia samples isolated from gorillas have been reported in humans, suggesting that the importance of humans in this ecosystem should be more closely evaluated.

Activation of the c-Met pathway occurs in a range of malignancies, including papillary renal cell carcinoma (RCC). Its activity in clear cell RCC is less clear. We investigated c-Met expression and inhibition in a large cohort of RCC tumors and cell lines.c-Met protein expression was determined by automated quantitative analysis (AQUA) on a tissue microarray (TMA) constructed from 330 RCC tumors paired with adjacent normal renal tissue. c-Met expression and selective inhibition with SU11274 and ARQ 197 were studied in clear cell RCC cell lines.Higher c-Met expression was detected in all RCC subtypes than in the adjacent normal renal tissue (P0.0001). Expression was highest in papillary and sarcomatoid subtypes, and high-grade and stage tumors. Higher c-Met expression correlated with worse disease-specific survival [risk ratio = 1.36; 95% confidence interval (CI) 1.08-1.74; P = 0.0091] and was an independent predictor of survival, maintained in clear cell subset analyses. c-Met protein was activated in all cell lines, and proliferation (and colony formation) was blocked by SU11274 and ARQ 197.c-Met is associated with poor pathologic features and prognosis in RCC. c-Met inhibition demonstrates in vitro activity against clear cell RCC. Further study of ARQ 197 with appropriate biomarker studies in RCC is warranted.

Chagas disease, which manifests as cardiomyopathy and severe gastrointestinal dysfunction, is caused by Trypanosoma cruzi, a vector-borne parasite. In California, the vector Triatoma protracta frequently colonizes woodrat (Neotoma spp.) lodges, but may also invade nearby residences, feeding upon humans and creating the dual risk of bite-induced anaphylaxis and T. cruzi transmission. Our research aimed to assess T. cruzi presence in woodrats in a previously unstudied northern California area, statistically evaluate woodrat microhabitat use with respect to vegetation parameters, and provide guidance for habitat modifications to mitigate public health risks associated with Tr. protracta exposure. Blood samples from big-eared woodrats (N. macrotis) trapped on rural private properties yielded a T. cruzi prevalence of 14.3%. Microhabitat analyses suggest that modifying vegetation to reduce understory density within a 40 meter radius of human residences might minimize woodrat lodge construction within this buffer area, potentially decreasing human exposure to Tr. protracta. Electronic supplementary material The online version of this article (doi:10.1007/s10393-016-1153-5) contains supplementary material, which is available to authorized users.

Rapidly developing coastal regions face consequences of land use and climate change including flooding and increased sediment, nutrient and chemical runoff, but these forces may also enhance pathogen runoff, which threatens human, animal and ecosystem health. Using the zoonotic parasite Toxoplasma gondii in California, USA as a model for coastal pathogen pollution, we examine the spatial distribution of parasite runoff and the impacts of precipitation and development on projected pathogen delivery to the ocean. Oocysts, the extremely hardy free-living environmental stage of T. gondii shed in faeces of domestic and wild felids, are carried to the ocean by freshwater runoff. Linking spatial pathogen loading and transport models, we show that watersheds with the highest levels of oocyst runoff align closely with regions of increased sentinel marine mammal T. gondii infection. These watersheds are characterized by higher levels of coastal development and larger domestic cat populations. Increases in coastal development and precipitation independently raised oocyst delivery to the ocean (average increases of 44% and 79%, respectively), but dramatically increased parasite runoff when combined (175% average increase). Anthropogenic changes in landscapes and climate can accelerate runoff of diverse pathogens from terrestrial to aquatic environments, influencing transmission to people, domestic animals and wildlife.

Tissue-cyst forming coccidia in the family Sarcocystidae are etiologic agents of protozoal encephalitis in marine mammals including the federally listed Southern sea otter (Enhydra lutris). California sea lions (Zalophus californianus), whose coastal habitat overlaps with sea otters, are definitive hosts for coccidian protozoa provisionally named Coccidia A, B and C. While Coccidia A and B have unknown clinical effects on aquatic wildlife hosts, Coccidia C is associated with severe protozoal disease in harbor seals (Phoca vitulina). In this study, we conducted surveillance for protozoal infection and fecal shedding in hospitalized and free-ranging California sea lions on the Pacific Coast and examined oocyst morphology and phenotypic characteristics of isolates via mouse bioassay and cell culture. Coccidia A and B were shed in similar frequency, particularly by yearlings. Oocysts shed by one free-ranging sea lion sampled at Año Nuevo State Park in California were previously unidentified in sea lions and were most similar to coccidia infecting Guadalupe fur seals (Arctocephalus townsendi) diagnosed with protozoal disease in Oregon (USA). Sporulated Coccidia A and B oocysts did not replicate in three strains of mice or in African green monkey kidney cells. However, cultivation experiments revealed that the inoculum of fecally-derived Coccidia A and B oocysts additionally contained organisms with genetic and antigenic similarity to Sarcocystis neurona; despite the absence of detectable free sporocysts in fecal samples by microscopic examination. In addition to the further characterization of Coccidia A and B in free-ranging and hospitalized sea lions, these results provide evidence of a new role for sea lions as putative mechanical vectors of S. neurona, or S. neurona-like species. Future work is needed to clarify the distribution, taxonomical status, and pathogenesis of these parasites in sea lions and other marine mammals that share their the near-shore marine environment., Graphical abstract, Highlights • Diverse coccidian protozoa shed by California sea lions (CSL) were characterized. • Oocyst shedding patterns, taxonomy, morphology and pathogenicity were examined. • Mice and cell cultures were not susceptible to Coccidia A or B of CSL origin. • Sarcocystis neurona-like zoites grew in cells inoculated with CSL fecal samples. • California sea lions may serve as mechanical vectors of an S. neurona-like organism.

Aquatic macroaggregates (flocs ≥ 0.5 mm) provide an important mechanism for vertical flux of nutrients and organic matter in aquatic ecosystems, yet their role in the transport and fate of zoonotic pathogens is largely unknown. Terrestrial pathogens that enter coastal waters through contaminated freshwater runoff may be especially prone to flocculation due to fluid dynamics and electrochemical changes that occur where fresh and marine waters mix. In this study, laboratory experiments were conducted to evaluate whether zoonotic pathogens (Cryptosporidium, Giardia, Salmonella) and a virus surrogate (PP7) are associated with aquatic macroaggregates and whether pathogen aggregation is enhanced in saline waters. Targeted microorganisms showed increased association with macroaggregates in estuarine and marine waters, as compared with an ultrapure water control and natural freshwater. Enrichment factor estimations demonstrated that pathogens are 2-4 orders of magnitude more concentrated in aggregates than in the estuarine and marine water surrounding the aggregates. Pathogen incorporation into aquatic macroaggregates may influence their transmission to susceptible hosts through settling and subsequent accumulation in zones where aggregation is greatest, as well as via enhanced uptake by invertebrates that serve as prey for marine animals or as seafood for humans.

Testicular teratomas result from anomalies in germ cell development during embryogenesis. In the 129 family of inbred strains of mice, teratomas initiate around embryonic day (E) 13.5 during the same developmental period in which female germ cells initiate meiosis and male germ cells enter mitotic arrest. Here, we report that three germ cell developmental abnormalities, namely continued proliferation, retention of pluripotency, and premature induction of differentiation, associate with teratoma susceptibility. Using mouse strains with low versus high teratoma incidence (129 versus 129-Chr19MOLF/Ei), and resistant to teratoma formation (FVB), we found that germ cell proliferation and expression of the pluripotency factor Nanog at a specific time point, E15.5, were directly related with increased tumor risk. Additionally, we discovered that genes expressed in pre-meiotic embryonic female and adult male germ cells, including cyclin D1 (Ccnd1) and stimulated by retinoic acid 8 (Stra8), were prematurely expressed in teratoma-susceptible germ cells and, in rare instances, induced entry into meiosis. As with Nanog, expression of differentiation-associated factors at a specific time point, E15.5, increased with tumor risk. Furthermore, Nanog and Ccnd1, genes with known roles in testicular cancer risk and tumorigenesis, respectively, were co-expressed in teratoma-susceptible germ cells and tumor stem cells, suggesting that retention of pluripotency and premature germ cell differentiation both contribute to tumorigenesis. Importantly, Stra8-deficient mice had an 88% decrease in teratoma incidence, providing direct evidence that premature initiation of the meiotic program contributes to tumorigenesis. These results show that deregulation of the mitotic-meiotic switch in XY germ cells contributes to teratoma initiation.

Routine fecal examination revealed novel coccidian oocysts in asymptomatic California sea lions (Zalophus californianus) in a rehabilitation facility. Coccidian oocysts were observed in fecal samples collected from 15 of 410 California sea lions admitted to The Marine Mammal Center between April 2007 and October 2009. Phylogenetic analysis using the full ITS-1 region, partial small subunit 18S rDNA sequence, and the Apicomplexa rpoB region identified 2 distinct sequence clades, referred to as Coccidia A and Coccidia B, and placed them in the Sarcocystidae, grouped with the tissue-cyst-forming coccidia. Both sequence clades resolved as individual taxa at ITS-1 and rpoB and were most closely related to Neospora caninum. Coccidia A was identified in 11 and Coccidia B in 4 of 12 sea lion oocyst samples successfully sequenced (3 of those sea lions were co-infected with both parasites). Shedding of Coccidia A oocysts was not associated with age class, sex, or stranding location, but yearlings represented the majority of shedders (8/15). This is the first study to use molecular phylogenetics to identify and describe coccidian parasites shed by a marine mammal.

The protozoan parasite Toxoplasma gondii infects a large proportion of threatened California sea otters (Enhydra lutris nereis), and is an important waterborne pathogen in humans. Contamination of coastal waters with T. gondii is thought to occur through delivery of environmentally resistant oocysts to nearshore regions via overland runoff. The objectives of this study were to evaluate whether T. gondii oocysts and surrogate microspheres attach to aggregates ($ 0.5 mm), and whether the magnitude of aggregation depends on water type, specifically salinity. Laboratory aggregation studies were conducted by adding T. gondii oocysts and surrogate microspheres to riverine, estuarine, and marine waters, and quantifying the proportion of oocysts and surrogates in aggregate-rich and aggregate-free water fractions. Attachment of oocysts and surrogates to aggregates occurred in all water types, but was greater in estuarine and marine waters, with concentrations of T. gondii in aggregates enriched 3–4 orders of magnitude. Aquatic aggregates may, thus, significantly influence waterborne transport of terrestrially derived pathogens, both through enhanced settling and subsequent concentration in the benthos, as well as by facilitating ingestion by invertebrate vectors that can transmit pathogens to susceptible hosts, including sea otters and humans.

The Toxoplasma gondii bradyzoite is essential to establish persistent infection, yet little is known about what factors this developmental form secretes to establish the cyst or interact with its host cell. To identify candidate bradyzoite-secreted effectors, the transcriptomes of in vitro tachyzoites 2 days postinfection, in vitro bradyzoites 4 days postinfection, and in vivo bradyzoites 21 days postinfection were interrogated by microarray, and the program SignalP was used to identify signal peptides indicating secretion. One hundred two putative bradyzoite-secreted effectors were identified by this approach. Two candidates, bradyzoite pseudokinase 1 and microneme adhesive repeat domain-containing protein 4, were chosen for further investigation and confirmed to be induced and secreted by bradyzoites in vitro and in vivo . Thus, we report the first analysis of the transcriptomes of in vitro and in vivo bradyzoites and identify two new protein components of the Toxoplasma tissue cyst wall.

Enteric protozoal infection was identified in 5 stranded California sea lions (Zalophus californianus). Microscopically, the apical cytoplasm of distal jejunal enterocytes contained multiple stages of coccidian parasites, including schizonts with merozoites and spherical gametocytes, which were morphologically similar to coccidians. By histopathology, organisms appeared to be confined to the intestine and accompanied by only mild enteritis. Using electron microscopy, both sexual (microgametocytes, macrogamonts) and asexual (schizonts, merozoites) coccidian stages were identified in enterocytes within parasitophorous vacuoles, consistent with apicomplexan development in a definitive host. Serology was negative for tissue cyst-forming coccidians, and immunohistochemistry for Toxoplasma gondii was inconclusive and negative for Neospora caninum and Sarcocystis neurona. Analysis of ITS-1 gene sequences amplified from frozen or formalin-fixed paraffin-embedded intestinal sections identified DNA sequences with closest homology to Neospora sp. (80%); these novel sequences were referred to as belonging to coccidian parasites ‘‘A,’’ ‘‘B,’’ and ‘‘C.’’ Subsequent molecular analyses completed on a neonatal harbor seal (Phoca vitulina) with protozoal lymphadenitis, hepatitis, myocarditis, and encephalitis showed that it was infected with a coccidian parasite bearing the ‘‘C’’ sequence type. Our results indicate that sea lions likely serve as definitive hosts for 3 newly described coccidian parasites, at least 1 of which is pathogenic in a marine mammal intermediate host species.

Northern sea otter (Enhydra lutris kenyoni) abundance has decreased dramatically over portions of southwest Alaska, USA, since the mid-1980s, and this stock is currently listed as threatened under the Endangered Species Act. In contrast, adjacent populations in south central Alaska, USA, and Russia have been stable to increasing during the same period. Sea otters bordering the area classified in the recent decline were live-captured during 2004-2006 at Bering Island, Russia, and the Kodiak Archipelago, Alaska, USA, to evaluate differences in general health and current exposure status to marine and terrestrial pathogens. Although body condition was lower in animals captured at Bering Island, Russia, than it was at Kodiak, USA, clinical pathology values did not reveal differences in general health between the two regions. Low prevalences of antibodies (5%) were found in Kodiak, USA, and on Bering Island, Russia, to Toxoplasma gondii, Sarcocystis neurona, and Leptospira interrogans. Exposure to phocine herpesvirus-1 was found in both Kodiak, USA (15.2%), and Bering Island, Russia (2.3%). Antibodies to Brucella spp. were found in 28% of the otters tested on Bering Island, Russia, compared with only 2.7% of the samples from Kodiak, USA. Prevalence of exposure to Phocine distemper virus (PDV) was 41% in Kodiak, USA, but 0% on Bering Island, Russia. Archived sera from southwest and south-central Alaska dating back to 1989 were negative for PDV, indicating exposure occurred in sea otters in Kodiak, USA, in recent years. Because PDV can be highly pathogenic in naïve and susceptible marine mammal populations, tissues should be examined to explore the contribution of this virus to otter deaths. Our results reveal an increase in exposure to pathogens in sea otters in Kodiak, Alaska, USA, since the 1990 s.

Purpose: Melanoma is relatively resistant to chemotherapy; improved targeting of molecules critical for cell proliferation and survival are needed. Phosphatidylinositol-3 kinase (PI3K) is an important target in melanoma; however, activity of PI3K inhibitors (PI3KI) is limited. Our purpose was to assess mTOR as a cotarget for PI3K. Methods: Using a method of quantitative immunofluorescence to measure mTOR expression in a large melanoma cohort, we studied associations with PI3K subunits, p85 and p110α. We assessed addition of the mTOR inhibitor rapamycin to 2 PI3KIs, NVP-BKM120 and LY294002. We studied in vitro activity of a novel dual PI3K/mTOR inhibitor NVP-BEZ235 and activity of the combination of NVP-BEZ235 and the MAP/ERK kinase (MEK) inhibitor AZD6244. Results: Strong coexpression of mTOR and p110α was observed (ρ = 0.658; P < 0.0001). Less coexpression was seen with p85 (ρ = 0.239; P < 0.0001). Strong synergism was shown between rapamycin and both PI3KIs. Activity of both PI3KIs was similarly enhanced with all rapamycin concentrations used. The dual PI3K/mTOR inhibitor effectively inhibited viability in 23 melanoma cell lines (IC50 values in the nanomolar range), regardless of B-Raf mutation status, with resultant reduction in clonogenicity and downregulation of pAkt and pP70S6K. Synergism was seen when combining NVP-BEZ235 and AZD6244, with resultant increases in poly(ADP-ribose) polymerase and caspase-2 cleavage. Conclusions: mTOR and p110α are coexpressed in melanoma. Rapamycin concentrations as low as 1 nmol/L enhance activity of PI3KIs. The dual PI3K/mTOR inhibitor NVP-BEZ235 is highly active in melanoma cells in vitro, suggesting that concurrent PI3K and mTOR targeting in melanoma warrants further investigation, both alone and in combination with MEK inhibitors.

The value of Bacteroidales genetic markers and fecal indicator bacteria (FIB) to predict the occurrence of waterborne pathogens was evaluated in ambient waters along the central California coast. Bacteroidales host-specific quantitative PCR (qPCR) was used to quantify fecal bacteria in water and provide insights into contributing host fecal sources. Over 140 surface water samples from 10 major rivers and estuaries within the Monterey Bay region were tested over 14 months with four Bacteroidales -specific assays (universal, human, dog, and cow), three FIB (total coliforms, fecal coliforms, and enterococci), two protozoal pathogens ( Cryptosporidium and Giardia spp.), and four bacterial pathogens ( Campylobacter spp., Escherichia coli O157:H7, Salmonella spp., and Vibrio spp.). Indicator and pathogen distribution was widespread, and detection was not highly seasonal. Vibrio cholerae was detected most frequently, followed by Giardia , Cryptosporidium , Salmonella , and Campylobacter spp. Bayesian conditional probability analysis was used to characterize the Bacteroidales performance assays, and the ratios of concentrations determined using host-specific and universal assays were used to show that fecal contamination from human sources was more common than livestock or dog sources in coastal study sites. Correlations were seen between some, but not all, indicator-pathogen combinations. The ability to predict pathogen occurrence in relation to indicator threshold cutoff levels was evaluated using a weighted measure that showed the universal Bacteroidales genetic marker to have a comparable or higher mean predictive potential than standard FIB. This predictive ability, in addition to the Bacteroidales assays providing information on contributing host fecal sources, supports using Bacteroidales assays in water quality monitoring programs.

To investigate how different routes of Toxoplasma gondii transmission influence the antibody response and infection status of deer mice (Peromyscus maniculatus), 80 mice were orally infected with 1, 5, 10, or 100 T. gondii oocysts. Ten weeks postinfection, 15 T. gondii -seropositive female mice were bred and allowed to produce 2 litters. Evidence of persistent T .gondii infection in orally infected mice was detected by serology and DNA amplification in mice from all 4 oocyst treatment groups, including those that received only a single T. gondii oocyst. Congenital transmission of T. gondii was detected by polymerase chain reaction (PCR) in 7/8 first and 4/7 second litters. Toxoplasma gondii was also detected by PCR in 9/30 congenitally infected offspring 16 wk after birth, despite the fact that detectable serological titers had waned. These findings raise questions about the applicability of serological testing to assess the prevalence of T. gondii infection in deer mice and other rodents in the wild. Additionally, the detection of frequent congenital transmission suggests that deer mice could help maintain T. gondii in the environment even in the absence of definitive feline hosts.

Sarcocystis neurona is a significant cause of neurological disease in horses and other animals, including the threatened Southern sea otter (Enhydra lutris nereis). Opossums (Didelphis virginiana), the only known definitive hosts for S. neurona in North America, are an introduced species in California. S. neurona DNA isolated from sporocysts and/or infected tissues of 10 opossums, 6 horses, 1 cat, 23 Southern sea otters, and 1 harbor porpoise (Phocoena phocoena) with natural infections was analyzed based on 15 genetic markers, including the first internal transcribed spacer (ITS-1) region; the 25/396 marker; S. neurona surface antigen genes (snSAGs) 2, 3, and 4; and 10 different microsatellites. Based on phylogenetic analysis, most of the S. neurona strains segregated into three genetically distinct groups. Additionally, fifteen S. neurona samples from opossums and several intermediate hosts, including sea otters and horses, were found to be genetically identical across all 15 genetic markers, indicating that fatal encephalitis in Southern sea otters and equine protozoal myeloencephalitis (EPM) in horses is strongly linked to S. neurona sporocysts shed by opossums.

The six mammalian CCN genes (Cyr61, CTGF, Nov, WISP1, WISP2, WISP3) encode a family of secreted, cysteine-rich, multimodular proteins having roles in cell proliferation, adhesion, migration, and differentiation during embryogenesis, wound healing, and angiogenesis. We used bioinformatics to identify 9 CCN genes in zebrafish (zCCNs), 6 of which have not been previously described. When compared with mammalian CCN family members, 3 were paralogs of Cyr61, 2 of CTGF, 2 of WISP1, 1 of WISP2, and 1 of WISP3. No paralog of Nov was found. In situ hybridization was performed to characterize the sites of expression of the zCCNs during early zebrafish development. zCCNs demonstrated both unique and overlapping patterns of expression, suggesting potential division of labor between orthologous genes and providing an alternate approach to gene function studies that will complement studies in mammalian models. Developmental Dynamics 239:1755–1767, 2010. © 2010 Wiley-Liss, Inc.

Toxoplasma gondii is a parasite of birds and mammals. Cats are the only definitive host and thus the only source of infective oocysts, but other mammals and birds can develop tissue cysts. Although feline infections are typically asymptomatic, infection during human pregnancy can cause severe disease in the fetus. Cat owners can reduce their pets' exposure risk by keeping all cats indoors and not feeding them raw meat. Humans usually become infected through ingestion of oocyst-contaminated soil and water, tissue cysts in undercooked meat, or congenitally. Because of their fastidious nature, the passing of non-infective oocysts, and the short duration of oocyst shedding, direct contact with cats is not thought to be a primary risk for human infection.

Three bald eagles ( Haliaeetus leucocephalus) and 1 golden eagle ( Aquila chrysaetos) were admitted to rehabilitation facilities with emaciation, lethargy, and an inability to fly. Intravascular schizonts and merozoites were present in 2 bald eagles, mainly in the lung tissue, whereas the third bald eagle and the golden eagle had lymphohistiocytic encephalitis with intralesional schizonts and merozoites. In all eagles, protozoal tissue cysts were present in skeletal musculature or heart. The protozoal organisms were morphologically compatible with a Sarcocystis sp. By immunohistochemistry, the protozoal merozoites were positive for Sarcocystis falcatula antigen in all cases when using polyclonal antisera. Furthermore, the protozoa were confirmed to be most similar to S. falcatula by polymerase chain reaction in 3 of the 4 cases. To the authors' knowledge, this report presents the first cases of natural infection in eagles with S. falcatula as a cause of mortality.

High use areas are a fundamental part of California coastal dairies and grazing livestock ranches as feeding areas, nurseries, and sick pens. High stocking densities and daily use in these areas lead to soil surfaces devoid of vegetation and covered in manure, with high potential for manure transport during winter rains to receiving waters regulated for shellfish harvesting and recreation. We characterized the association between California's Mediterranean climate and a series of existing and proposed management practices on fecal coliform bacteria (FCB) transport from high use areas on dairies and ranches. Results from 351 storm runoff samples collected below 35 high-use areas indicate that removal of cattle during winter, locating high use areas on level ground, application of straw and seeding, and vegetative buffer strip implementation were significantly associated with FCB concentration and load reductions. These results complement our findings for reductions of specific pathogens in runoff from these areas. These findings have practical significance because they document surface water quality benefits that the studied management practices provide in application on working farms and ranches. This direction is critical and timely for on-farm management efforts seeking to reduce microbial pollution in runoff and comply with indicator bacteria water quality criteria.

In 2004, three wild sea otters were diagnosed with putative Sarcocystis neurona -associated meningoencephalitis by histopathology and immunohistochemistry. Schizonts, free merozoites and tissue cysts were observed in the brains of all three infected animals. Tissue cysts walls from sea otter 1 (SO1) stained positively using anti- S. neurona polyclonal antiserum. However, positive staining does not preclude infection by closely related or cross-reactive tissue cyst-forming coccidian parasites. Two immature tissue cysts in the brain of SO1 were examined using transmission electron microscopy. Ultrastructural features included cyst walls with thin villous projections up to 1 μm long with tapered ends and a distinctive, electron-dense outer lining layer composed of linearly-arranged, semi-circular structures with a “hobnailed” surface contour. Small numbers of microtubules extended down through the villi into the underlying granular layer. Metrocytes were short and plump with an anterior apical complex, 22 sub-pellicular microtubules, numerous free ribosomes and no rhoptries. Some metrocytes appeared to be dividing, with two adjacent nuclear profiles. Collectively these ultrastructural features were compatible with developing protozoal cysts and were similar to prior descriptions of S. neurona tissue cysts. Panspecific 18S rDNA primers were utilized to identify protozoa infecting the brains of these otters and DNA amplification and additional sequencing at the ITS1 locus confirmed that all three otters were infected with S. neurona . No other Sarcocystis spp. were detected in the brains or skeletal muscles of these animals by immunohistochemistry or PCR. We believe this is the first ultrastructural and molecular confirmation of the development of S. neurona tissue cysts in the CNS of any animal.

The processes promoting disease in wild animal populations are highly complex, yet identifying these processes is critically important for conservation when disease is limiting a population. By combining field studies with epidemiologic tools, we evaluated the relationship between key factors impeding southern sea otter ( Enhydra lutris nereis ) population growth: disease and resource limitation. This threatened population has struggled to recover despite protection, so we followed radio-tagged sea otters and evaluated infection with 2 disease-causing protozoal pathogens, Toxoplasma gondii and Sarcocystis neurona , to reveal risks that increased the likelihood of pathogen exposure. We identified patterns of pathogen infection that are linked to individual animal behavior, prey choice, and habitat use. We detected a high-risk spatial cluster of S. neurona infections in otters with home ranges in southern Monterey Bay and a coastal segment near San Simeon and Cambria where otters had high levels of infection with T. gondii . We found that otters feeding on abalone, which is the preferred prey in a resource-abundant marine ecosystem, had a very low risk of infection with either pathogen, whereas otters consuming small marine snails were more likely to be infected with T. gondii . Individual dietary specialization in sea otters is an adaptive mechanism for coping with limited food resources along central coastal California. High levels of infection with protozoal pathogens may be an adverse consequence of dietary specialization in this threatened species, with both depleted resources and disease working synergistically to limit recovery.

The physical properties that govern the waterborne transmission of Toxoplasma gondii oocysts from land to sea were evaluated and compared to the properties of carboxylated microspheres, which could serve as surrogates for T. gondii oocysts in transport and water treatment studies. The electrophoretic mobilities of T. gondii oocysts, lightly carboxylated Dragon Green microspheres, and heavily carboxylated Glacial Blue microspheres were determined in ultrapure water, artificial freshwater with and without dissolved organic carbon, artificial estuarine water, and artificial seawater. The surface wettabilities of oocysts and microspheres were determined using a water contact angle approach. Toxoplasma gondii oocysts and microspheres were negatively charged in freshwater solutions, but their charges were neutralized in estuarine water and seawater. Oocysts, Glacial Blue microspheres, and unwashed Dragon Green microspheres had low contact angles, indicating that they were hydrophilic; however, once washed, Dragon Green microspheres became markedly hydrophobic. The hydrophilic nature and negative charge of T. gondii oocysts in freshwater could facilitate widespread contamination of waterways. The loss of charge observed in saline waters may lead to flocculation and subsequent accumulation of T. gondii oocysts in locations where freshwater and marine water mix, indicating a high risk of exposure for humans and wildlife in estuarine habitats with this zoonotic pathogen. While microspheres did not have surface properties identical to those of T. gondii , similar properties shared between each microsphere type and oocysts suggest that their joint application in transport and fate studies could provide a range of transport potentials in which oocysts are likely to behave.

Feces of harbor seals (Phoca vitulina richardsi) and hybrid glaucous-winged/western gulls (Larus glaucescens / occidentalis) from Washington State's inland marine waters were examined for Giardia and Cryptosporidium spp. to determine if genotypes carried by these wildlife species were the same genotypes that commonly infect humans and domestic animals. Using immunomagnetic separation followed by direct fluorescent antibody detection, Giardia spp. cysts were detected in 42% of seal fecal samples (41/97). Giardia-positive samples came from 90% of the sites (9/10) and the prevalence of positive seal fecal samples differed significantly among study sites. Fecal samples collected from seal haulout sites with over 400 animals were 4.7 times more likely to have Giardia spp. cysts than samples collected at smaller haulout sites. In gulls, a single Giardia sp. cyst was detected in 4% of fecal samples (3/78). Cryptosporidium spp. oocysts were not detected in any of the seals or gulls tested. Sequence analysis of a 398 bp segment of G. duodenalis DNA at the glutamate dehydrogenase locus suggested that 11 isolates originating from seals throughout the region were a novel genotype and 3 isolates obtained from a single site in south Puget Sound were the G. duodenalis canine genotype D. Real-time TaqMan PCR amplification and subsequent sequencing of a 52 bp small subunit ribosomal DNA region from novel harbor seal genotype isolates showed sequence homology to canine genotypes C and D. Sequence analysis of the 52 bp small subunit ribosomal DNA products from the 3 canine genotype isolates from seals produced mixed sequences at could not be evaluated.

Objectives. The goal of this study was to identify potential environmental and demographic factors associated with Campylobacter jejuni (C. jejuni), Salmonella enterica (Salmonella spp.), and antimicrobial-resistant Escherichia coli (E. coli) infection in northern elephant seals stranded along the California coastline. Methods. E. coli, Salmonella spp., and C. jejuni were isolated from rectal swabs from 196 juvenile northern elephant seals, which were found stranded and alive along the California coast and brought to The Marine Mammal Center in Sausalito, California, for rehabilitation. Gender, weight, county where the animal stranded, month stranded, coastal human population density, exposure to sewage outfall or freshwater outflow (river or stream), and cumulative precipitation in the previous 24 hours, seven days, 30 days, 90 days, and 180 days were analyzed as potential risk factors for infection. Results. The odds of C. jejuni and antimicrobial-resistant E. coli were higher in feces of seals stranded at sites with higher levels of freshwater outflow compared with lower levels of freshwater outflow. The odds of Salmonella spp. in feces were 5.4 times greater in seals stranded in locations with lower levels of 30-day cumulative precipitation, along with substantially lower odds of Salmonella shedding for seals stranded in Monterey or Santa Cruz county compared with seals stranded in regions further north or south of this central California location. Conclusions. Juvenile northern elephant seals that have entered the water are being colonized by antimicrobial-resistant and pathogenic fecal bacteria that may be acquired from terrestrial sources transmitted via river and surface waters.