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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.

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.

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.

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.

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.

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.

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.

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 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.

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.

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.

Toxoplasma gondii, a ubiquitous parasitic protozoan, is emerging as an aquatic biological pollutant. Infections can result from drinking water contaminated with environmentally resistant oocysts. However, recommendations regarding water treatment for oocyst inactivation have not been established. In this study, the physical method of radiofrequency (RF) power was evaluated for its ability to inactivate T. gondii oocysts in water. Oocysts were exposed to various RF energy levels to induce 50, 55, 60, 70 and 80 degrees C temperatures maintained for 1 min. Post-treatment oocyst viability was determined by mouse bioassay with serology, immunohistochemistry and in vitro parasite isolation to confirm T. gondii infections in mice. None of the mice inoculated with oocysts treated with RF-induced temperatures of > or =60 degrees C in an initial experiment became infected; however, there was incomplete oocyst activation in subsequent experiments conducted under similar conditions. These results indicate that T. gondii oocysts may not always be inactivated when exposed to a minimum of 60 degrees C for 1 min. The impact of factors such as water heating time, cooling time and the volume of water treated must be considered when evaluating the efficacy of RF power for oocyst inactivation.

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 aim of this study was to determine if antimicrobial drug use increases resistance of commensal gastrointensinal Escherichia coli of wild northern elephant seals ( Mirounga angustirostris ) treated in rehabilitation, and, if so, identify the risk factors involved. Minimum inhibitory concentration (MIC) levels of twelve antimicrobial drugs were determined for 289 E. coli isolates from 99 seals sampled at admission and 277 isolates obtained at release from rehabilitation using broth microdilution. Prevalence of E. coli antimicrobial resistance, MIC 50 , MIC 90 , and clustering of MIC values were determined for seals and the data were analyzed using Fisher's exact test, ordinal logistic regression and negative binomial regression. At release from rehabilitation 77.8% of the seals had antimicrobial resistant E. coli compared to 38.4% of the seals at admission. The MIC 90 for amoxicillin-clavulanic acid, chloramphenicol, enrofloxacin, ticarcillin-clavulanic acid, and trimethoprim-saulfamethoxazole were at levels considered to be sensitive at admission but they increased to levels of resistance at release. E. coli were grouped into four clusters by their MIC values, with increasing levels of resistance going from Cluster 1 to 4. A primary risk factor associated with the probability of a seal having E. coli in Clusters 3 and 4 was time in rehabilitation, regardless of whether the animal received treatment with antimicrobial drugs, suggesting nosocomial infection. The results of this study provide evidence that increased levels of hygiene and appropriate use of antimicrobial therapy might be important in the rehabilitation of wild animals to prevent rise in the prevalence of antimicrobial resistant bacteria.

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.

Objective—To estimate the analytic sensitivity of microscopic detection of Toxoplasma gondii oocysts and the environmental loading of T gondii oocysts on the basis of prevalence of shedding by owned and unowned cats. Design—Cross-sectional survey. Sample Population—326 fecal samples from cats. Procedures—Fecal samples were collected from cat shelters, veterinary clinics, cat-owning households, and outdoor locations and tested via ZnSO4 fecal flotation. Results—Only 3 (0.9%) samples of feces from 326 cats in the Morro Bay area of California contained T gondii–like oocysts. On the basis of the estimated tonnage of cat feces deposited outdoors in this area, the annual burden in the environment was estimated to be 94 to 4,671 oocysts/m2 (9 to 434 oocysts/ft2). Conclusions and Clinical Relevance—Despite the low prevalence and short duration of T gondii oocyst shedding by cats detected in the present and former surveys, the sheer numbers of oocysts shed by cats during initial infection could lead to substantial environmental contamination. Veterinarians may wish to make cat owners aware of the potential threats to human and wildlife health posed by cats permitted to defecate outdoors.

Eight female Peromyscus californicus were infected with 10(2) or 10(4) Toxoplasma gondii culture-derived tachyzoites (Type II or X) isolated from southern sea otters. All but 2 mice survived infection and developed antibodies to T. gondii. The 2 fatally infected mice were inoculated with 10(4) tachyzoites of the Type X strain. Parasite detection by immunohistochemistry (IHC) and DNA amplification with 2 polymerase chain reaction (PCR) methods was compared for brain, heart, lung, liver, spleen, biceps muscle, and tongue, at a mean of 41 days postinfection. Parasites were detected most commonly by IHC in spleen (8/8) and brain (6/8). DNA amplification by PCR was most successful from brain, heart, and spleen.

We evaluated the sensitivity (Se) and specificity (Sp) of an IgG enzyme-linked immunosorbent assay (ELISA) and IgG indirect fluorescent antibody test (IFAT) for detection of Toxoplasma gondii-specific antibodies in sera from 2 cat populations using a Bayesian approach. Accounting for test covariance, the Se and Sp of the IgG ELISA were estimated to be 92.6% and 96.5%, and those of the IgG IFAT were 81.0% and 93.8%, respectively. Both tests performed poorly in cats experimentally coinfected with feline immunodeficiency virus and T. gondii. Excluding this group, Se and Sp of the ELISA were virtually unchanged (92.3% and 96.4%, respectively), whereas the IFAT Se improved to 94.2% and Sp remained stable at 93.7%. These tests and an IgM ELISA were applied to 123 cat sera from the Morro Bay area, California, where high morbidity and mortality attributable to toxoplasmosis have been detected in southern sea otters. Age-adjusted IgG seroprevalence in this population was estimated to be 29.6%, and it did not differ between owned and unowned cats. Accounting for Se, Sp, and test covariances, age-adjusted seroprevalence was 45.0%. The odds for T. gondii seropositivity were 12.3-fold higher for cats aged12 mo compared with cats aged6 mo.

The purpose of this study was to determine the effect of blood contamination of cerebrospinal fluid (CSF) on the results of indirect fluorescent antibody tests (IFATs) for Sarcocystis neurona and Neospora hughesi. The in vitro study used antibody-negative CSF collected from non-neurologic horses immediately after euthanasia and blood samples from 40 healthy horses that had a range of IFAT antibody titers against S. neurona and N. hughesi. Serial dilutions of whole blood were made in seronegative CSF to generate blood-contaminated CSF with red blood cell (RBC) concentrations ranging from 10 to 100,000 RBCs/μl. The blood-contaminated CSF samples were then tested for antibodies against both pathogens using IFAT. Blood contamination of CSF had no detectable effect on IFAT results for S. neurona or N. hughesi at any serologic titer when the RBC concentration in CSF was

There are only two reports in the literature demonstrating the presence of Campylobacter spp. in marine mammals. One report describes the isolation of a new species, Campylobacter insulaenigrae sp. nov., from three harbor seals ( Phoca vitulina ) and a harbor porpoise ( Phocoena phocoena ) in Scotland, and the other describes the isolation of Campylobacter jejuni , Campylobacter lari , and an unknown Campylobacter species from northern elephant seals ( Mirounga angustirostris ) in California. In this study, 72 presumptive C. lari and unknown Campylobacter species strains were characterized using standard phenotypic methods, 16S rRNA PCR, and multilocus sequence typing (MLST). Phenotypic characterization of these isolates showed them to be variable in their ability to grow either at 42°C or on agar containing 1% glycine and in their sensitivity to nalidixic acid and cephalothin. Based on both 16S rRNA PCR and MLST, all but 1 of the 72 isolates were C. insulaenigrae , with one isolate being similar to but distinct from both Campylobacter upsaliensis and Campylobacter helveticus . Phylogenetic analysis identified two C. insulaenigrae clades: the primary clade, containing exclusively California strains, and a secondary clade, containing some California strains and all of the original Scottish strains. This study demonstrates the inability of phenotypic characterization to correctly identify all Campylobacter species and emphasizes the importance of molecular characterization via 16S rRNA sequence analysis or MLST for the identification of Campylobacter isolates from marine mammals.

The investigation of diseases of free-ranging river otters (Lontra canadensis) is a primary conservation priority for this species; however, very little is known about diseases of river otters that forage in marine environments. To identify and better understand pathogens that could be important to marine-foraging river otters, other wildlife species, domestic animals, and humans and to determine if proximity to human population could be a factor in disease exposure, serum samples from 55 free-ranging marine-foraging river otters were tested for antibodies to selected pathogens. Thirty-five animals were captured in Prince William Sound, Alaska (USA), an area of low human density, and 20 were captured in the San Juan Islands, Washington State (USA), an area characterized by higher human density. Of 40 river otters tested by indirect immunofluorescent antibody test, 17.5% were seropositive (titeror =320) for Toxoplasma gondii. All positive animals came from Washington. Of 35 river otters tested for antibodies to Leptospira interrogans using the microscopic agglutination test, 10 of 20 (50%) from Washington were seropositive (titeror =200). None of the 15 tested animals from Alaska were positive. Antibodies to Neospora caninum (n=40), Sarcocystis neurona (n=40), Brucella abortus (n=55), avian influenza (n=40), canine distemper virus (n=55), phocine distemper virus (n=55), dolphin morbillivirus (n=55), porpoise morbillivirus (n=55), and Aleutian disease parvovirus (n=46) were not detected. Identifying exposure to T. gondii and L. interrogans in otters from Washington State but not in otters from Alaska suggests that living proximal to higher human density and its associated agricultural activities, domestic animals, and rodent populations could enhance river otter exposure to these pathogens.

SUMMARYToxoplasma gondiiandSarcocystis neuronaare protozoan parasites with terrestrial definitive hosts, and both pathogens can cause fatal disease in a wide range of marine animals. Close monitoring of threatened southern sea otters (Enhydra lutris nereis) in California allowed for the diagnosis of dual transplacental transmission ofT. gondiiandS. neuronain a wild female otter that was chronically infected with both parasites. Congenital infection resulted in late-term abortion due to disseminated toxoplasmosis.Toxoplasma gondiiandS. neuronaDNA was amplified from placental tissue culture, as well as from fetal lung tissue. Molecular characterization ofT. gondiirevealed a Type X genotype in isolates derived from placenta and fetal brain, as well as in all tested fetal organs (brain, lung, spleen, liver and thymus). This report provides the first evidence for transplacental transmission ofT. gondiiin a chronically infected wild sea otter, and the first molecular and immunohistochemical confirmation of concurrent transplacental transmission ofT. gondiiandS. neuronain any species. Repeated fetal and/or neonatal losses in the sea otter dam also suggested thatT. gondiihas the potential to reduce fecundity in chronically infected marine mammals through parasite recrudescence and repeated fetal infection.

Recent reports of Sarcocystis fayeri-induced toxicity in people consuming horse meat warrant investigation on the prevalence and molecular characterization of Sarcocystis spp. infection in horses. Sarcocysts in skeletal muscle of horses have been commonly regarded as an incidental finding. In this study, we investigated the prevalence of sarcocysts in skeletal muscle of horses with neuromuscular disease. Our findings indicated that S. fayeri infection was common in young mature horses with neuromuscular disease and could be associated with myopathic and neurogenic processes. The number of infected muscles and number of sarcocysts per muscle were significantly higher in diseased than in control horses. S. fayeri was predominantly found in low oxidative highly glycolytic myofibers. This pathogen had a high glycolytic metabolism. Common clinical signs of disease included muscle atrophy, weakness with or without apparent muscle pain, gait deficits, and dysphagia in horses with involvement of the tongue and esophagus. Horses with myositis were lethargic, apparently painful, stiff, and reluctant to move. Similar to humans, sarcocystosis and cardiomyopathy can occur in horses. This study did not establish causality but supported a possible association (8.9% of cases) with disease. The assumption of Sarcocysts spp. being an incidental finding in every case might be inaccurate.

This study was designed to determine the relative levels of gene transcription of selected pathogens and cytokines in the brain and spinal cord of 12 horses with equine protozoal myeloencephalitis (EPM), 11 with equine herpesvirus type 1 (EHV-1) myeloencephalopathy, and 12 healthy control horses by applying a real time pcr to the formalin-fixed and paraffin-embedded tissues. Total rna was extracted from each tissue, transcribed to complementary dna (cDNA) and assayed for Sarcocystis neurona, Neospora hughesi, EHV-1, equine GAPDH (housekeeping gene), tumour necrosis factor (TNF)-alpha, interferon (IFN)-gamma, interleukin (IL)-1beta, IL-2, IL-4, IL-6, IL-8, IL-10 AND IL-12 p40. S neurona cdna was detected in the neural tissue from all 12 horses with EPM, and two of them also had amplifiable cDNA of N hughesi. The relative levels of transcription of protozoal cdna ranged from 1 to 461 times baseline (mean 123). All the horses with ehv-1 myeloencephalopathy had positive viral signals by PCR with relative levels of transcription ranging from 1 to 1618 times baseline (mean 275). All the control horses tested negative for S neurona, N hughesi and EHV-1 cdna. The cytokine profiles of each disease indicated a balance between pro- and anti-inflammatory markers. In the horses with epm the pro-inflammatory Th1 cytokines (IL-8, TNF-alpha and IFN-gamma) were commonly expressed but the anti-inflammatory Th2 cytokines (IL-4, IL-6 AND IL-10) were absent or rare. In the horses with ehv-1 the proinflammatory cytokine IL-8 was commonly expressed, but IL-10 and IFN-gamma were not, and TNF-alpha was rare. Tissue from the control horses expressed only the gene GAPDH.

Objective—To determine gene transcription for cytokines in nucleated cells in CSF of horses without neurologic signs or with cervical stenotic myelopathy (CSM), West Nile virus (WNV) encephalitis, equine protozoal myeloencephalitis (EPM), or spinal cord trauma.Animals—41 horses (no neurologic signs [n = 12], CSM [8], WNV encephalitis [9], EPM [6], and spinal cord trauma [6]).Procedures—Total RNA was extracted from nucleated cells and converted into cDNA. Gene expression was measured by use of real-time PCR assay and final quantitation via the comparative threshold cycle method.Results—Cytokine genes expressed by nucleated cells of horses without neurologic signs comprised a balance between proinflammatory tumor necrosis factor-α (TNF-α), anti-inflammatory cytokines (interleukin [IL]-10 and transforming growth factor [TGF]-β), and Th1 mediators (interferon [IFN]-γ). Cells of horses with CSM mainly expressed genes for TNF-α, TGF-β, and IL-10. Cells of horses with WNV encephalitis mainly expressed genes for IL-6 and TGF-β. Cells of horses with EPM mainly had expression of genes for IL-6, IL-8, IL-10, TNF-α, IFN-γ, and TGF-β. Cells from horses with spinal cord trauma had expression mainly for IL-6; IFN-γ; TGF-β; and less frequently, IL-2, IL-10, and TNF-α. Interleukin-8 gene expression was only detected in CSF of horses with infectious diseases.Conclusions and Clinical Relevance—Despite the small number of CSF samples for each group, results suggest distinct gene signatures expressed by nucleated cells in the CSF of horses without neurologic signs versus horses with inflammatory or traumatic neurologic disorders.

The morphologic, ultrastructural and genotypic characteristics of Babesia duncani n.sp. are described based on the characterization of two isolates (WA1, CA5) obtained from infected human patients in Washington and California. The intraerythrocytic stages of the parasite are morphologically indistinguishable from Babesia microti, which is the most commonly identified cause of human babesiosis in the USA. Intraerythrocytic trophozoites of B. duncani n.sp. are round to oval, with some piriform, ring and ameboid forms. Division occurs by intraerythrocytic schizogony, which results in the formation of merozoites in tetrads (syn. Maltese cross or quadruplet forms). The ultrastructural features of trophozoites and merozoites are similar to those described for B. microti and Theileria spp. However, intralymphocytic schizont stages characteristic of Theileria spp. have not been observed in infected humans. In phylogenetic analyses based on sequence data for the complete18S ribosomal RNA gene, B. duncani n.sp. lies in a distinct clade that includes isolates from humans, dogs and wildlife in the western United States but separate from Babesia sensu stricto, Theileria spp. and B. microti. ITS2 sequence analysis of the B. duncani n.sp. isolates (WA1, CA5) show that they are phylogenetically indistinguishable from each other and from two other human B. duncani-type parasites (CA6, WA2 clone1) but distinct from other Babesia and Theileria species sequenced. This analysis provides robust molecular support that the B. duncani n.sp. isolates are monophyletic and the same species. The morphologic characteristics together with the phylogenetic analysis of two genetic loci support the assertion that B. duncani n.sp. is a distinct species from other known Babesia spp. for which morphologic and sequence information are available.

This collection of abstracts provides an account of four presentations at the 19th International Conference of the World Association for the Advancement of Veterinary Parasitology (WAAVP) (held in New Orleans, LA, USA from 10-14 August 2003) in a symposium session on zoonotic protozoan parasites found in the marine environment and chaired by Ronald Fayer and David Lindsay. The focus was on three genera of parasites of veterinary and public health concern-Toxoplasma, Giardia, and Cryplosporidium with emphasis on their epidemiology in the marine environment.

Detailed postmortem examination of southern sea otters (Enhydra lutris nereis) found along the California (USA) coast has provided an exceptional opportunity to understand factors influencing survival in this threatened marine mammal species. In order to evaluate recent trends in causes of mortality, the demographic and geographic distribution of causes of death in freshly deceased beachcast sea otters necropsied from 1998-2001 were evaluated. Protozoal en- cephalitis, acanthocephalan-related disease, shark attack, and cardiac disease were identified as common causes of death in sea otters examined. While infection with acanthocephalan parasites was more likely to cause death in juvenile otters, Toxoplasma gondii encephalitis, shark attack, and cardiac disease were more common in prime-aged adult otters. Cardiac disease is a newly recognized cause of mortality in sea otters and T. gondii encephalitis was significantly associated with this condition. Otters with fatal shark bites were over three times more likely to have pre- existing T. gondii encephalitis suggesting that shark attack, which is a long-recognized source of mortality in otters, may be coupled with a recently recognized disease in otters. Spatial clusters of cause-specific mortality were detected for T. gondii encephalitis (in Estero Bay), acanthoceph- alan peritonitis (in southern Monterey Bay), and shark attack (from Santa Cruz to Point Ano Nuevo). Diseases caused by parasites, bacteria, or fungi and diseases without a specified etiology were the primary cause of death in 63.8% of otters examined. Parasitic disease alone caused death in 38.1% of otters examined. This pattern of mortality, observed predominantly in juvenile and prime-aged adult southern sea otters, has negative implications for the overall health and recovery of this population.

A serum indirect fluorescent antibody test (IFAT) was compared with a Western blot (WB) and a modified Western blot (mWB) for diagnosis of equine protozoal myeloencephalitis (EPM). Using receiver-operating characteristic (ROC) analysis, the area under the curve of the IFAT was greater than the area under the curves of the WB and the mWB ( P = 0.025 and P = 0.044, respectively). There was no statistically significant difference between the areas under the curves of the WBs ( P > 0.05). On the basis of an arbitrarily chosen cut-off titer for a positive test result of 1:80 for the IFAT and interpreting weak positive WB results as positive test results, the sensitivities and 95% confidence intervals (CI) of all 3 tests were identical and equal to 88.9% (51.8–99.7%). The specificities and 95% CIs of the IFAT, WB, and mWB test were 100% (91–100%), 87.2% (72.6–95.7%), and 69.2% (52.4–83%), respectively. The overall accuracy of the IFAT was shown to be better than that of the WBs and, therefore, the test has potential for use in the diagnosis of EPM caused by Sarcocystis neurona.

Neospora hughesi is a newly recognized protozoan pathogen in horses that causes a myeloencephalitis similar to Sarcocystis neurona. There are no validated serologic tests using the gold standard sera that are currently available to detect specific N. hughesi antibodies and, thus, no tests available to detect antemortem exposure or estimate seroprevalence in the horse. The objectives of the present study were to establish a bank of gold standard equine sera through experimental infections with N. hughesi and to assess several serologic tests for the detection of related protozoan antibodies. Seven horses were inoculated with N. hughesi tachyzoites, and 7 horses received uninfected cell culture material. The horses were monitored, and blood and cerebrospinal fluid were collected repeatedly over a 4-mo period. With the sera, 4 different serologic techniques were evaluated, including a whole-parasite lysate enzyme-linked immunosorbent assay (ELISA), a recombinant protein ELISA, a modified direct agglutination test, and an indirect fluorescent antibody test. Qualitative and quantitative evaluation of the results showed that the N. hughesi indirect fluorescent antibody test (IFAT) consistently discriminated between experimentally infected and noninfected horses, using a cutoff of 1:640. Sera from 3 naturally infected horses had titers >1:640. Cerebrospinal fluid in all but 1 infected horse had very low N. hughesi IFAT titers (

Developing an effective vaccine against neosporosis presents several interesting challenges. The parasite is spread efficiently from mother to foetus over several generations, and naturally infected cattle do not appear to develop adequate protective immunity. Modulation of the immune response during pregnancy favours parasite survival and multiplication. However, induction of pro-inflammatory responses that are thought to be protective against Neospora caninum would be detrimental to the pregnancy. So, is vaccination a feasible option to control the disease? This article discusses some of these issues and reports on the progress towards a vaccine for neosporosis.

Neosporosis is an economically important disease of dairy cattle caused by the protozoanNeospora caninum. Diagnostic tests for neosporosis are complicated by the potential for cross-reaction of antibodies to antigens that are similar betweenN. caninumand closely related parasitesToxoplasma gondiiandSarcocystis cruzi.To provide a sensitive and specific assay for detecting antibodies toN. caninumin the serum of infected animals, we have investigated a recombinant form of the antigen known as Ncp29 (rNcp29), which is a major surface protein of the parasite. Ncp29 is encoded by a gene that is homologous to theSAG1gene previously characterized fromT. gondii.An enzyme-linked immunosorbent assay (ELISA) was used to screen animals for the presence of serum antibodies specific to rNcp29. The rNcp29 ELISA readily distinguished between cattle known to be infected withN. caninum(optical density [OD] > 1.2 at 1:500 or greater dilution) and negative controls (OD < 0.5 at 1:500). Additionally, sera from animals that were infected withT. gondiiorS. cruziwere negative. The rNcp29 ELISA developed here provides a specific and sensitive assay for detecting neosporosis in cattle.

This study evaluates whether medical and veterinary students' attitudes toward "One Health" and interprofessional education changed after participating in a joint small group learning exercise focused on risk factors associated with zoonotic disease. A survey was distributed to third-year medical students (n = 98) and second-year veterinary students (n = 140), each with a 95% response rate. Overall, 92% of veterinary students and 73% of medical students agreed or strongly agreed that "One Health" was relevant to their desired specialty. Students from both schools largely agreed that interprofessional education should be a goal of the curriculum for their school, and that interprofessional approaches strengthen their overall education. Students reported increased confidence in their communication skills and improved ability to contribute to One Health collaborative teams. This educational intervention, built around a patient case, focused on a variety of learning objectives including skills (such as communication), knowledge (of zoonotic toxoplasmosis) and attitudes (toward collaborative learning and practice). By sparking an interest in One Health during their early professional education, we sought to encourage a new generation of physicians and veterinarians to adopt a more collaborative spirit to their clinical practice, which will ultimately benefit human, animal and environmental health.