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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

Physicians and veterinarians are increasingly expected to collaborate across disciplines; however, in most cases their education and training remain isolated within their respective professions. Medical and veterinary students are rarely provided with opportunities for inter-professional learning during their coursework and clinical training. One Health serves as an ideal framework for developing problem-focused curricula that promote inter-professional teamwork. One Health issues (e.g., zoonotic diseases, water pollution, toxic waste, impact of climate change, and food safety and security) not only engage students across disciplines, but require faculty and senior leadership across various health-related fields to share knowledge and balance perspectives throughout curriculum development and implementation. In this article, we report on one of several interactive, small-group, case-based One Health curricular exercises developed collaboratively by students and faculty in our Schools of Medicine and Veterinary Medicine to ensure that all students, regardless of background or intended specialty, would receive a basic introduction to inter-professional collaboration in the context of a One Health clinical problem of the sort they might encounter in their future practice. Toxoplasmosis ( Toxoplasma gondii infection) was selected as the first case because of the potentially different perspectives that medical and veterinary practitioners may have on advising a pregnant woman with regard to risk factors, prevention, testing, and treatment. Our goal was to develop an evidence-based approach to this clinical case that could be used by both professions to assess environmental and zoonotic risk factors for T. gondii in human pregnancies.

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.

Costa Rica undertakes continuous efforts to recover the native population of macaw species through rehabilitation programs for breeding and releasing birds in protected areas. In the summer of 2018, a total of 107 scarlet (

Human babesiosis is a tick-borne protozoal disease of increasing clinical significance in North America. Most cases in the eastern and Midwestern regions of the United States are reportedly due to Babesia microti infections. By contrast, most human infections reported in California and Washington have been attributed to a new species that was first identified in 1991 and subsequently named Babesia duncani. Although the tick vector and mammalian reservoir hosts for B. microti are well characterized, the vector and reservoir hosts for B. duncani are unknown. As a result, specific risk factors for human infections cannot be characterized. Identification of potential hosts and vector species has been hampered by the lack of specific and sensitive molecular diagnostic tools to amplify parasite DNA. To address this need, a nested PCR assay targeting the β-tubulin gene, a well-conserved locus in piroplasm parasites with a highly variable intron region among species, was developed. The assay was evaluated by spiking tick and mammalian DNA extracts with DNA from a B. duncani isolate derived from a human patient (WA-1) as well as related Babesia spp. from Californian wildlife. This assay was highly specific, with a sensitivity of approximately 1 copy of template DNA in a background of tick DNA. At this level of detection B. duncani was detectable in larval tick samples, and the target locus allowed for visual differentiation between species by gel electrophoresis. This assay offers researchers a new tool for elucidating the natural transmission cycle of B. duncani.

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 recognizes the interconnectedness of human, animal, and ecosystem health and encourages collaboration between diverse disciplines to address complex health problems. In this paper, 3 academics, with diverse training, experience and backgrounds who each work on different pathogenic parasites, will share their stories of tackling parasitic challenges by applying a One Health approach. The pathogenic parasites to be discussed include the helminth Taenia solium and protozoans Giardia, Theileria, Babesia, Neospora and Toxoplasma species. The 3 narratives focus on research and clinical case-based challenges and illustrate where collaboration between human, animal, and environmental health scientists either has or could lead to improved control of human and animal health as well as important research discoveries. The need for better evaluation of interventions and scientific evidence to support changes in clinical practice and encourage enhanced collaboration between human and veterinary clinicians, as well as new governmental policies to improve public and wildlife health, are described. The need for a range of evidence-based metrics to monitor the success and impact of the One Health approach to veterinary parasitology is also discussed.

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.

An ante-mortem diagnosis of equine protozoal myeloencephalitis (EPM) is presently based on clinical presentation, immunodiagnostics performed on serum and cerebrospinal fluid (CSF), and ruling out other neurological disorders. Molecular techniques introduce a novel and promising approach for the detection of protozoal agents in CSF. Hypothesizing that real-time PCR (rtPCR) can be a useful complement to EPM diagnostics, 210 CSF samples from horses suspected of neurological disease with EPM included as a differential diagnosis were tested using rtPCR to detect Sarcocystis neurona DNA and immunodiagnostics targeting antibodies against the same pathogen, performed on serum and CSF samples. Molecular and immunological results were compared with respect to origin of the horse, time of the year, signalment, clinical signs and treatment history. Twenty-five horses tested positive in CSF for S. neurona by rtPCR only, while 30 horses had intrathecally-derived antibodies to S. neurona only (serum to CSF ratio ≤ 64 by indirect fluorescent antibody test - IFAT), and 13 horses tested rtPCR-positive in CSF with evidence of intrathecally-derived antibodies to S. neurona. Previous treatment for EPM was the only variable presenting statistical difference between the two testing modalities, highlighting that animals with history of anti-protozoal treatment were more likely to test positive solely in IFAT, while horses without treatment were more likely to test positive by rtPCR only. The results support the use of molecular diagnosis for EPM caused by S. neurona as a complement to immunodiagnostics. The use of rtPCR in CSF for the detection of S. neurona may improve the diagnostic work-up of neurologic disease suspected horses, especially in animals without previous anti-protozoal treatment.

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.

SUMMARYSmall marine snails and abalone have been identified as high- and low-risk prey items, respectively, for exposure of threatened southern sea otters to Toxoplasma gondii, a zoonotic parasite that can cause fatal encephalitis in animals and humans. While recent work has characterized snails as paratenic hosts for T. gondii, the ability of abalone to vector the parasite has not been evaluated. To further elucidate why abalone predation may be protective against T. gondii exposure, this study aimed to determine whether: (1) abalone are physiologically capable of acquiring T. gondii; and (2) abalone and snails differ in their ability to concentrate and retain the parasite. Abalone were exposed to T. gondii surrogate microspheres for 24 h, and fecal samples were examined for 2 weeks following exposure. Concentration of surrogates was 2–3 orders of magnitude greater in abalone feces than in the spiked seawater, and excretion of surrogates continued for 14 days post-exposure. These results indicate that, physiologically, abalone and snails can equally vector T. gondii as paratenic hosts. Reduced risk of T. gondii infection in abalone-specializing otters may therefore result from abalone's high nutritional value, which implies otters must consume fewer animals to meet their caloric needs.

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.

SUMMARYSarcocystis neurona is a terrestrial parasite that can cause fatal encephalitis in the endangered Southern sea otter (Enhydra lutris nereis). To date, neither risk factors associated with marine contamination nor the route of S. neurona infection to marine mammals has been described. This study evaluated coastal S. neurona contamination using California mussels (Mytilus californianus) as sentinels for pathogen pollution. A field investigation was designed to test the hypotheses that (1) mussels can serve as sentinels for S. neurona contamination, and (2) S. neurona contamination in mussels would be highest during the rainy season and in mussels collected near freshwater. Initial validation of molecular assays through sporocyst spiking experiments revealed the ITS-1500 assay to be most sensitive for detection of S. neurona, consistently yielding parasite amplification at concentrations ⩾5 sporocysts/1 mL mussel haemolymph. Assays were then applied on 959 wild-caught mussels, with detection of S. neurona confirmed using sequence analysis in three mussels. Validated molecular assays for S. neurona detection in mussels provide a novel toolset for investigating marine contamination with this parasite, while confirmation of S. neurona in wild mussels suggests that uptake by invertebrates may serve as a route of transmission to susceptible marine animals.

Costa Rica undertakes continuous efforts to recover the native population of macaw species through rehabilitation programs for breeding and releasing birds in protected areas. In the summer of 2018, a total of 107 scarlet (Ara macao) and 93 great green (Ara ambigua) macaws were sampled in four wildlife rehabilitation centers in Costa Rica. Fecal samples representing 200 individuals were analyzed for intestinal parasites, and 23 individuals were sampled for hemoparasites. Ascaridia and Capillaria were found in fecal samples. No hemoparasites were found. The distribution of percentage of infection was analyzed by location, species, and housing type. As part of a health screening prior to release, parasitological examination is recommended.

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.

Summary The parasite Toxoplasma gondii is an environmentally persistent pathogen that can cause fatal disease in humans, terrestrial warm-blooded animals and aquatic mammals. Although an association between T. gondii exposure and prey specialization on marine snails was identified in threatened California sea otters, the ability of kelp-dwelling snails to transmit terrestrially derived pathogens has not been previously investigated. The objective of this study was to measure concentration and retention of T. gondii by marine snails in laboratory aquaria, and to test for natural T. gondii contamination in field-collected snails. Following exposure to T. gondii-containing seawater, oocysts were detected by microscopy in snail faeces and tissues for 10 and 3 days respectively. Nested polymerase chain reaction was also applied as a method for confirming putative T. gondii oocysts detected in snail faeces and tissues by microscopy. Toxoplasma gondii was not detected in field-collected snails. Results suggest that turban snails are competent transport hosts for T. gondii. By concentrating oocysts in faecal pellets, snails may facilitate entry of T. gondii into the nearshore marine food web. This novel mechanism also represents a general pathway by which marine transmission of terrestrially derived microorganisms can be mediated via pathogen concentration and retention by benthic invertebrates.

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).

Endemic zoonoses, such as Q fever and spotted fever group (SFG) rickettsiosis, are prevalent in South Africa, yet often undiagnosed. In this study, we reviewed the demographics and animal exposure history of patients presenting with acute febrile illness to community health clinics in Mpumalanga Province to identify trends and risk factors associated with exposure to Coxiella burnetii, the causative agent of Q fever, and infection by SFG Rickettsia spp. Clinical and serological data and questionnaires elucidating exposure to animals and their products were obtained from 141 acutely febrile patients between 2012 and 2016. Exposure or infection status to C. burnetii and SFG Rickettsia spp. was determined by presence of IgG or IgM antibodies. Logistic regression models were built for risk factor analysis. Clinical presentation of patients infected by SFG rickettsiosis was described. There were 37/139 (27%) patients with a positive C. burnetii serology, indicative of Q fever exposure. Patients who had reported attending cattle inspection facilities ("dip tanks") were 9.39 times more likely to be exposed to Q fever (95% CI: 2.9-30.4). Exposure risk also increased with age (OR: 1.03, 95% CI: 1.002-1.06). Twenty-one per cent of febrile patients (24/118) had evidence of acute infection by SFG Rickettsia spp. Similarly, attending cattle inspection facilities was the most significant risk factor (OR: 8.48, 95% CI: 1.58-45.60). Seropositivity of females showed a significant OR of 8.0 when compared to males (95% CI: 1.49-43.0), and consumption of livestock was associated with a decreased risk (OR: 0.02, 95% CI: 0.001-0.54). A trend between domestic cat contact and SFG rickettsiosis was also noted, albeit borderline non-significant. In this endemic region of South Africa, an understanding of risk factors for zoonotic pathogens, including exposure to domestic animals, can help clinic staff with diagnosis and appropriate therapeutic management of acutely febrile patients as well as identify target areas for education and prevention strategies.

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.

Summary Coastal habitat contamination with Toxoplasma gondii is a health risk to humans and marine wildlife, with infections documented in both nearshore and pelagic marine mammals. Due to lack of sensitive methods for detection of T. gondii in water, this study utilized an alternative surveillance approach for evaluating marine habitat contamination using wild mussels. The objectives of this study were to (i) validate sensitive molecular tools for T. gondii detection in mussels and (ii) apply optimized methods in a surveillance study to determine the prevalence and genotype(s) of T. gondii in mussels. Simplex polymerase chain reaction screening and multiplex genotyping assays were validated and then applied on 959 wild-caught mussels collected from central California. Thirteen mussels (1.4%) had detectable T. gondii DNA and the presence of T. gondii in mussels was significantly associated with proximity to freshwater run-off and collection during the wet season. Molecular characterization revealed alleles from T. gondii types I, II/III, X at the B1 locus, and a novel atypical B1 allele that was recently documented in T. gondii-infected carnivores from California. Findings demonstrate higher than previously reported T. gondii contamination of California coastlines, and describe novel strains of the parasite that further link terrestrial sources with marine contamination.

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.

Cryptosporidium parvum, Giardia lamblia, and Toxoplasma gondii are waterborne protozoal pathogens distributed worldwide and empirical evidence suggests that wetlands reduce the concentrations of these pathogens under certain environmental conditions. The goal of this study was to evaluate how protozoal removal in surface water is affected by the water temperature, turbidity, salinity, and vegetation cover of wetlands in the Monterey Bay region of California. To examine how protozoal removal was affected by these environmental factors, we conducted observational experiments at three primary spatial scales: settling columns, recirculating wetland mesocosm tanks, and an experimental research wetland (Molera Wetland). Simultaneously, we developed a protozoal transport model for surface water to simulate the settling columns, the mesocosm tanks, and the Molera Wetland. With a high degree of uncertainty expected in the model predictions and field observations, we developed the model within a Bayesian statistical framework. We found protozoal removal increased when water flowed through vegetation, and with higher levels of turbidity, salinity, and temperature. Protozoal removal in surface water was maximized (~0.1 hour(-1)) when flowing through emergent vegetation at 2% cover, and with a vegetation contact time of ~30 minutes compared to the effects of temperature, salinity, and turbidity. Our studies revealed that an increase in vegetated wetland area, with water moving through vegetation, would likely improve regional water quality through the reduction of fecal protozoal pathogen loads.

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.

The One Health approach, which recognizes the interconnectedness of human, animal and ecosystem health, encourages collaboration between diverse disciplines to address complex health problems. The advantages and challenges posed by these interdisciplinary collaborations are described in this review. Learning networks where diverse participants can openly share processes, best practices, and case studies are discussed as a strategy for conducting transdisciplinary One Health research and tackling complex global health problems. The 11 papers in this special issue are also introduced as they illustrate how a One Health approach can be applied to better understand and control zoonotic pathogens, engage community stakeholders in One Health research and utilize wildlife species, most notably sea otters and birds, as sentinels of ecosystem health. Collaboration is rarely without complications; however, drawing on these insights may benefit the process of operationalizing the One Health approach to address today's global health challenges.

Livestock fairs present a unique opportunity for the public to experience close contact with animals, but may also expose people to zoonotic pathogens through contact with animal feces. The goal of this study was to screen cattle, sheep, goat, chicken, rabbit and horse feces from a livestock fair in California for the potentially zoonotic pathogens Escherichia coli O157:H7, Salmonella, Campylobacter, Vibrio, Cryptosporidium and Giardia spp., as well as determining the level of antimicrobial resistance in E. coli and Salmonella. Notably, E. coli O157:H7 was reported for the first time in a pig at a county fair in California. Campylobacter jejuni as well as Salmonella enterica serovars Derby and Thompson were also isolated from pigs, cattle, sheep, goats or chickens, whereas horses and rabbits were negative for all target pathogens. The prevalence of antimicrobial resistance as well as multi-drug resistance patterns were highest for E. coli and Salmonella spp. cultured from pigs and chickens, were generally widespread but at lower levels for other animal groups, and included resistance to ampicillin and streptomycin, two antimicrobial drugs of importance for human medicine. This study provides data that highlight the importance of practicing good hygiene in livestock fair settings to avoid transmission of zoonotic microbes, particularly pathogens with antimicrobial resistance, to fair visitors and among animal populations.

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.

While toxoplasmosis is not commonly considered a clinical disease of equines, previous seroprevalence studies have reported differing background rates of Toxoplasma gondii infection in horses globally. The objective of this study was to evaluate possible associations between T. gondii seroprevalence and clinical signs of equine protozoal myeloencephalitis (EPM) in horses. Using a case-control study design, 720 Californian horses with neurologic signs compatible with EPM were compared to healthy, non-neurologic horses for the presence of T. gondii antibodies (using indirect fluorescent antibody tests [IFAT]). Toxoplasma gondii seroprevalence among cases and controls was determined at standard serum cut-offs: 40, 80, 160, 320, and 640. At a T. gondii titre cut-off of 320, horses with clinical signs compatible with EPM had 3.55 times the odds of a seropositive test compared to those without clinical signs (P

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.