Strategies and checklist for designing and conducting palliative care research with family carers: EAPC international expert elicitation study.

BACKGROUND: Palliative care services seek to improve the wellbeing of family carers of people living with serious and life-limiting illness. To help achieve this goal, systematic reviews have recommended priority areas for family carer research and the need to improve the quality of study design. Policy makers have also advocated for enhanced family carer support. However, there are specific methodological considerations and challenges in designing and conducting carer research conducted during the course of the serious illness trajectory and in bereavement. AIM: To develop strategies to improve the design and conduct of research with family carers. DESIGN: Expert elicitation study using an adapted version of the 'Identify, Discuss, Estimate and Aggregate' elicitation protocol, supplemented with strategies from peer-reviewed literature. SETTING/PARTICIPANTS: Nine members of the management committee of the European Association for Palliative Care's Reference group on family carer research, comprising international senior research academics in family caregiving. RESULTS: A compilation of recommended strategies and checklist was created to: (a) help researchers plan research involving family carers focussing on: preparation, conduct and dissemination and (b) assist ethics committees and funding bodies to evaluate proposals. CONCLUSIONS: The strategies and checklist for conducting research with family carers may enhance methodologically rigorous research. Consequently, researchers, practitioners and policy makers will not only gain a more comprehensive understanding of the unmet needs of family carers but also promote the development of empirically sound interventions.

Prioritisation of areas for early detection of southward movement of arctic fox rabies based on historical surveillance data in Quebec, Canada.

Arctic rabies virus variant (ARVV) is enzootic in Quebec (Canada) north of the 55th parallel. With climate change, increased risk of re-incursion of ARVV in more densely populated southern regions raises public and animal health concerns. The objective of this study was to prioritise geographical areas to target for an early detection of ARVV incursion south of the 55th parallel based on the historical spatio-temporal trends of reported rabies in foxes in Quebec. Descriptive analyses of fox rabies cases from 1953 to 2017 were conducted. Three periods show increases in the number of fox rabies cases in southern regions and indicate incursion from northern areas or neighbouring provinces. The available data, particularly in central and northern regions of the province, were scarce and of low spatial resolution, making it impossible to identify the path of spread with precision. Hence, we investigated the use of multiple criteria, such as historical rabies cases, human population density and red fox (Vulpes vulpes) relative abundance, to prioritise areas for enhanced surveillance. This study underscores the need to define and maintain new criteria for selecting samples to be analysed in order to detect rapidly ARVV cases outside the current enzootic area and any potential re-incursion of the virus into central and southern regions of the province.

First report of Gongylonema sp. in a free ranging callitrichid from the Brazilian Atlantic Forest: case report / Primeiro relato de Gongylonema sp. em um calitriquídeo de vida livre proveniente da Mata Atlântica brasileira: relato de caso

This is the first report of parasitism by Gongylonema sp. in a free-ranging callitrichid from the Atlantic forest of Espírito Santo, Brazil. A juvenile male of Geoffroy's marmoset (Callithrix geoffroyi) was euthanized due to poor prognosis, then necropsied. Samples of the tongue were collected for routine histological processing. Microscopically, there were transversal sections of adult nematodes within the epithelial layer of the mucosa of the tongue. Lingual scraping demonstrated a small number of oval embryonated eggs with a thick capsule. The morphology of the adult parasites and the eggs, associated with its location, were compatible with the Spirurida nematode of the genus Gongylonema. Further studies are needed to evaluate the impact of this parasite on free-ranging callitrichid populations.(AU)

Este é o primeiro relato de parasitismo por Gongylonema sp. em um calitriquídeo de vida livre proveniente da Mata Atlântica do Espírito Santo, Brasil. Um sagui-da-cara-branca (Callithrix geoffroyi), macho, jovem, foi eutanasiado, devido a prognóstico desfavorável, e necropsiado. Fragmentos de língua foram coletados para processamento histológico de rotina. Microscopicamente, havia cortes transversais de nematoides adultos na mucosa da língua. Na raspagem da mucosa da língua, foi observada pequena quantidade de ovos larvados ovais com cápsula espessa. A morfologia do parasita adulto e dos ovos, associada à localização do agente, é compatível com nematoide Spirurida do gênero Gongylonema. São necessários estudos adicionais para avaliar o impacto desse parasito nas populações de calitriquídeos de vida livre.(AU)

Schistosomes and Microfilarial Parasites in Magellanic Penguins.

The Magellanic Penguin ( Spheniscus magellanicus) is native to Argentina, Chile, and the Falkland/Malvinas Islands, and is a regular winter migrant in Uruguayan and Brazilian coastal waters. The species is known to be susceptible to a variety of gastrointestinal nematodes, cestodes, trematodes, and acanthocephalans, as well as renal trematodes and pulmonary nematodes. Schistosomes (Platyhelminthes, Trematoda, Schistosomatidae) and microfilariae (Nematoda, Secernentea, Onchocercidae) were histologically identified in Magellanic Penguins ( Spheniscus magellanicus) that died while under care at rehabilitation centers in southern Brazil. Phylogenetic analysis of the COI gene, ITS-1 region, 5.8S rRNA gene, ITS-2 region, and 28S rRNA gene sequences of the schistosome revealed that it is closely related to, but distinct from, a schistosome reported from the African Penguin ( Spheniscus demersus). The schistosomes from Magellanic and African Penguins were grouped with Gigantobilharzia huronensis, Gigantobilharzia melanoidis, and Dendritobilharzia pulvurenta; however, the lack of a clearly monophyletic origin precludes determining their genus. The incidental discovery of novel parasites during a study that did not specifically aim to investigate the occurrence of helminths underscores the value of histopathological examination as an exploratory diagnostic approach.

Rabies control in North America - past, present and future.

In the first half of the 20th century, rabies in dogs affected Canada, Mexico and the United States of America (USA). However, the role of wildlife in the transmission of the rabies virus was also recognised and outbreaks affecting both wildlife and domestic animals were documented. Canine rabies has since been eliminated from Canada and the USA, and is now only found sporadically in a few southern states of Mexico. Wildlife variant rabies viruses, found throughout the continent and geographically associated with specific reservoir species, have notable public and animal health, as well as economic, impacts. Early rabies control efforts included legislated dog management strategies and wildlife population reduction, which met with varying success. In the last 30 years, programmes for the control of rabies in dogs and wildlife have benefited from an 'Integrated Management Approach', combining education, vaccination (parenteral and oral), strategic population control, responsible pet ownership and effective stewardship of natural resources, in addition to cooperation and collaboration among local, national and international stakeholders. Looking ahead, the goal of eliminating specific wildlife virus variants will be challenged by the potential range expansion of reservoir species, due to climate change and other factors, and the risk of re-introducing eliminated virus variants. To be successful, programmes must be sustained and accompanied by advances in vaccines, enhanced distribution strategies, monitoring in the field and effective modelling of disease spread. They should also be informed by robust case surveillance, phylogenetics and an increased knowledge of vector ecology.

Au Canada, au Mexique et aux États-Unis d'Amérique, la rage canine a sévi jusqu'au milieu du xxe siècle. Le rôle de la faune sauvage dans la transmission du virus de la rage y était également connu et des foyers affectant aussi bien des espèces sauvages que domestiques étaient observés et étudiés. Depuis, le Canada et les États-Unis ont réussi à éliminer la rage canine de leur territoire, de sorte que cette maladie ne se déclare plus que sous forme sporadique dans certains états du Sud du Mexique. En revanche, les variants des virus de la rage qui affectent la faune sauvage circulent sur tout le territoire continental, leur présence étant géographiquement associée à celle de certaines espèces déterminées qui font office de réservoirs, avec des conséquences importantes pour la santé tant publique qu'animale et pour l'économie. Les premières mesures de lutte appliquées contre la rage, essentiellement des stratégies de contrôle des populations de chiens à travers une législation appropriée et une réduction de certaines populations d'animaux sauvages, ont rencontré un succès variable. Depuis une trentaine d'années, les programmes de lutte contre la rage chez les chiens et les animaux sauvages font l'objet d'une « démarche de gestion intégrée ¼ associant des campagnes de sensibilisation, l'application de la vaccination (par voies parentérale et orale), la maîtrise stratégique des populations, la responsabilisation des propriétaires de chiens et la gestion efficace des ressources naturelles, en plus de la coopération et de la collaboration entre les parties prenantes à l'échelle locale, nationale et internationale. En ce qui concerne l'avenir, l'objectif d'éliminer les variants du virus affectant spécifiquement la faune sauvage se heurte à l'expansion potentielle du spectre d'espèces réservoirs à la faveur du changement climatique et d'autres facteurs, ainsi qu'au risque de réintroduction de variants du virus précédemment éliminés. Pour réussir, les programmes doivent être soutenus dans le temps et s'accompagner d'avancées en matière de vaccins, de stratégies de distribution renforcées, d'un meilleur suivi sur le terrain et d'une modélisation efficace de la propagation de la maladie. Ils doivent également reposer sur une surveillance robuste des cas, sur des données phylogénétiques et sur une connaissance accrue de l'écologie des vecteurs.

En la primera mitad del siglo XX, la rabia estaba presente en la población canina del Canadá, México y los Estados Unidos de América, aunque también se conocía la función de los animales silvestres en la transmisión del virus y se habían descrito brotes que afectaron a la vez a animales domésticos y silvestres. Posteriormente la rabia canina fue eliminada del Canadá y los EE.UU. mientras que en México, a día de hoy, solo hace apariciones esporádicas en unos pocos estados meridionales. Las variantes del virus rábico que afectan a la fauna silvestre, presentes en todo el continente y geográficamente ligadas a determinadas especies que actúan de reservorio, tienen notables repercusiones en la salud pública, la sanidad animal y la economía. Entre las primeras iniciativas de lucha contra la rabia había medidas de reducción de las poblaciones de animales silvestres y estrategias de gestión de la población canina impuestas por vía legislativa que se aplicaron con éxito variable. En los últimos 30 años, los programas de lucha antirrábica en perros y animales silvestres han incorporado métodos de «gestión integrada¼ que aúnan labores de pedagogía, vacunación (parenteral y oral), control estratégico de poblaciones, propiedad responsable de los animales de compañía y eficaz administración de los recursos naturales, además de la cooperación y colaboración entre los interlocutores locales, nacionales e internacionales. De cara al futuro, el objetivo de eliminar variantes víricas específicas de la fauna silvestre se verá comprometido por el posible crecimiento del área de distribución de las especies reservorio (debido al cambio climático y otros factores) y por el riesgo de reintroducción de variantes víricas eliminadas. Para que los programas tengan éxito deben ser duraderos y acompañarse de avances en las vacunas, mejores estrategias de distribución, tareas de seguimiento sobre el terreno y elaboración de modelos eficaces de diseminación de la enfermedad. También deben alimentarse de una sólida vigilancia de casos, estudios filogenéticos y un mejor conocimiento de la ecología de los vectores.

Raccoon rabies outbreak in Hamilton, Ontario: A progress report.

BACKGROUND: Raccoon rabies is caused by a variant of the rabies virus found in raccoons but transmissible to other mammalian species, including humans. The disease of rabies caused by raccoon variant rabies virus is indistinguishable from rabies caused by other rabies virus variants. OBJECTIVE: This paper describes the raccoon rabies outbreak in Ontario (identified in December 2015) and the control measures undertaken to curb the spread of the epizootic using the One Health approach. INVESTIGATION AND RESULTS: Representatives from local, provincial and federal agencies collectively activated a raccoon rabies response that involved policy updates, enhanced surveillance, a public education campaign and mass vaccination of wildlife and domestic animals. Between December 2015 and June 2017, 338 animals tested positive for raccoon rabies in Ontario. While the majority of the cases were raccoons, there was significant spillover into striped skunks, as well as other species including two cats, a fox and a llama. Viral genome sequencing determined that this epizootic was likely caused by long-distance translocation from the United States. CONCLUSION: This outbreak of raccoon rabies is by far the largest to have occurred in Canada and the first raccoon rabies outbreak documented in a densely populated urban area. This is also the first time this rabies virus variant has been identified in a domestic animal in Canada. A collaborative approach involving numerous stakeholders in the public and private sectors has been instrumental in addressing this epizootic. Though case incidence appears to be declining, several years will likely be required to reach elimination. Continued collaboration between these agencies is necessary to achieve this goal.

A performance evaluation of a novel human recombinant tissue factor prothrombin time reagent (Revohem™ PT).

INTRODUCTION: A new prothrombin time reagent (Revohem™ PT) based on recombinant human tissue factor produced by the silkworm-baculovirus expression system was tested. The aim of this study was to compare the performance of the new PT reagent with two widely used routine PT reagents. METHODS: All testing was performed on a Sysmex CS-5100 coagulometer. Revohem™ PT was tested for imprecision and stability using normal and abnormal lyophilized commercial control plasmas. Comparability was assessed with two widely used reagents: one containing recombinant human tissue factor (Reagent A) and the other a human placental thromboplastin (Reagent B) using a wide range of normal and abnormal plasmas and analyser-specific ISI values. RESULTS: Excellent between-day imprecision was obtained for Revohem™ PT (CV <1.0%) and acceptable open-vial on-board stability over 7 days. There was good agreement between methods in samples from patients with liver disease and patients receiving warfarin and no significant differences between methods with increasing INR values. Both recombinant reagents suffered less interference from lupus anticoagulant than the placental thromboplastin. Revohem™ PT had similar sensitivity to reagents A and B for FII, V, VII and X deficiency and demonstrated dose responsiveness to dabigatran, apixaban and rivaroxaban with steeper response curves than the comparison reagents. CONCLUSION: Revohem™ PT showed comparable or improved performance relative to two widely used reagents and is suitable for use in warfarin control, detection of inherited factor II, V, VII and X deficiency and assessment of liver disease coagulopathy.

Hypovitaminosis A in extensively grazed beef cattle.

CASE REPORT: Vitamin A deficiency was diagnosed in a herd of 97 19-month-old Braford heifers in the Mitchell Grass Downs (Astrebla spp.) bioregion of Hughenden in north-western Queensland during November 2015. Two heifers died after a 48-h history of sternal recumbency and of the 19 that had neurological signs, 7 were blind. Histological changes in the optic nerves of the two necropsied cattle were consistent with vitamin A deficiency. This diagnosis was supported by vitamin A concentrations in fresh liver samples of 5 and 6 mg/kg wet tissue (reference range, 100-175 mg/kg) despite treatment of the cattle with twice the recommended dose of parenteral vitamin A 3 weeks prior to sampling. Rainfall on the property during the 2 years before the outbreak was less than the annual rainfall average of 464 mm, with a total of 281 mm in 2014 and 117 mm from January to November in 2015, most of this falling in January. CONCLUSION: Plant assays for both ß-carotene and crude protein concentrations in dry matter (DM) were less than the recommended dietary requirements for beef cattle (0.30 mg/kg DM and 56 g/kg, respectively).

Natural killer cells from psoriasis vulgaris patients have reduced levels of cytotoxicity associated degranulation and cytokine production.

Psoriasis vulgaris is a chronic inflammatory disease of the skin with a strong genetic component and immune system involvement. Although some evidence suggests that Natural Killer (NK) cells may play a part in psoriasis, their role is relatively unstudied and results are controversial. In this current study, NK cells from psoriasis patients exhibited reduced degranulation and produced lower levels of the pro-inflammatory cytokines IFN-γ and TNF-α. Further investigation found that NK cells from psoriasis patients and healthy controls expressed similar levels of activation markers, NK cell receptors and apoptosis-inducing molecules. In addition, comparable levels of several cytokines important in NK cell biology were found in the serum of psoriasis patients and healthy controls. Genotyping analysis revealed that HLA-C2, which provides a ligand for killer-cell immunoglobulin-like receptors (KIR) expressed by NK cells, was strongly associated with psoriasis susceptibility. However, no link between the KIR genes themselves and disease was found.

Successful Example of How to Implement and Develop a Deceased Organ Donation System in the Caribbean Region: Five-Year Experience of the SEUSA Program in Trinidad and Tobago.

BACKGROUND: The SEUSA program, the Donation and Transplantation Institute foundation consultancy program, was implemented in Trinidad and Tobago (T&T) in 2010 with the support of the National Organ Transplant Unit (NOTU) and the Ministry of Health of T&T. METHODS: The SEUSA program included (1) diagnosis of the current situation using the ODDS (Organ Donation Diagnostic Surveys); (2) creation of a human resources structure through Transplant Procurement Management (TPM); (3) detection of all brain and cardiac deaths in the hospitals implementing the DAS (Decease Alert System); (4) in-hospital awareness based on the EODS (Essentials in Organ Donation); and (5) external hospital audits. Additionally continued monitoring is performed. RESULTS: Thus far, thanks to implementation of the SEUSA program in Trinidad and Tobago 175, healthcare professionals have been exposed to training programs in the organ donation field. The Living Kidney Program was reinforced and the structure of the Deceased Donation (DD) network was defined. Since 2010, 485 potential organ donors have been detected, and 9 have become actual organ donors; 74 patients have received a kidney transplant (59 from living and 15 from deceased donors). CONCLUSIONS: This project results demonstrate that the application of the SEUSA program is an efficient methodology to develop DD programs that increase and consolidate transplant programs in the Caribbean region.

Increased frequencies of the killer immunoglobulin-like receptor genes KIR2DL2 and KIR2DS2 are associated with neuroblastoma.

Neuroblastoma is the most common extra-cranial solid tumour in children. Natural killer (NK) cells are innate lymphocytes that are known to mediate the direct cytotoxicity of neuroblastoma tumour cells. Natural variation in the highly polymorphic killer immunoglobulin-like receptors (KIR) and their cognate human leukocyte antigen (HLA) class I ligands results in considerable diversity in NK cell function. As the early onset of neuroblastoma suggests the contribution of genetic factors, we investigated if individual KIR genes, combined KIR gene haplotypes or compound KIR-HLA ligand genotypes could influence susceptibility to neuroblastoma. Genotype analysis of the KIR genes as well as their three major HLA class I ligand groups, HLA-C1, HLA-C2 and HLA-Bw4, was carried out in a cohort of 201 neuroblastoma patients compared with 240 healthy control subjects using polymerase chain reaction with sequence-specific primers. We found a significant increase in the frequency of KIR2DL2 (P = 0.019) as well as KIR2DS2 (P = 0.008) in patients with neuroblastoma compared with the healthy control group. While the incidence of the least inhibitory compound KIR-HLA-C genotype, KIR2DL3 in the presence of HLA-C1 was slightly reduced in neuroblastoma patients, this did not reach statistical significance (P = 0.069). In summary, while KIR-HLA compound genotypes have previously been implicated in predicting treatment outcomes in neuroblastoma, here we show that the presence of the individual KIR genes, KIR2DL2 and KIR2DS2, irrespective of HLA-C genotype is associated with the onset of this embryonal malignancy.

2DL1, 2DL2 and 2DL3 all contribute to KIR phenotype variability on human NK cells.

Natural killer (NK) cells are lymphocytes that function as part of the innate immune system. Their activity is controlled by a range of inhibitory and activating receptors, including the important killer-cell immunoglobulin-like receptors (KIR). The KIR are a multi-gene family of receptors that interact with the human leukocyte antigen (HLA) class I family of molecules and are characterised by extensive allelic polymorphism. Their expression on the cell surface of NK cells is highly variable, but the factors responsible for this variability are not yet clearly understood. In the current study, we investigated KIR expression in a healthy human cohort that we had previously characterised in depth at a genetic level, with KIR allele typing and HLA class I ligand genotypes available for all donors (n=198). Allelic polymorphism significantly affected the phenotypic expression of all KIR analysed, whereas HLA ligand background influenced the expression levels of 2DL1 and 2DL3. In particular, we found that although 2DL2 may influence 2DL1 expression, this appears to be owing to variation in 2DL1 copy number. Finally, the inhibitory receptor LILRB1 had higher expression levels in individuals with B/B KIR genotypes, suggesting a possible relationship between KIR and non-KIR receptors, which serves to balance NK cell activation potential.

Oral vaccination and protection of striped skunks (Mephitis mephitis) against rabies using ONRAB®.

Skunks are one of the most important rabies vector species in North America due to their wide geographic distribution, high susceptibility to the rabies virus, and tendency to inhabit areas around human dwellings and domestic animals. Oral vaccination is a cost-effective, socially acceptable technique often used to control rabies in terrestrial wildlife; however, control of rabies in skunks has proven especially challenging due to the lack of a vaccine effective by the oral route in this species. In this study, we examined the antibody response of captive striped skunks (Mephitis mephitis) to ONRAB(®) and tested the protection afforded by the vaccine against rabies virus. Thirty-one skunks were each offered one ONRAB(®) vaccine bait, 25 skunks were administered ONRAB(®) via direct instillation into the oral cavity (DIOC) and ten controls received no vaccine. A blood sample was collected from controls and vaccinates 6 weeks prior to treatment, and then 5 and 7 weeks post-vaccination (PV). A competitive ELISA was used to detect rabies antibody (RAb). Pre-vaccination sera for all skunks, and sera for all controls throughout the serology study, were negative for RAb. Fifty-eight percent (18/31) of skunks in the bait group and 100% (25/25) of skunks that received ONRAB(®) DIOC had detectable RAb by 7 week PV. All 10 controls succumbed to experimental rabies infection. In the group of skunks administered ONRAB(®) DIOC, 100% (23/23) survived challenge 247 days PV. Survival of skunks presented ONRAB(®) baits was 81% (25/31). In the bait group, all 18 skunks that had detectable RAb by 7 week PV survived challenge. Seven additional skunks without detectable RAb prior to week 7 PV also survived. Lack of any remarkable pathology in study animals, together with positive serology and challenge results, supports that ONRAB(®) is a safe and effective oral rabies vaccine for use in skunks.

Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing.

BACKGROUND: Enumeration of extracellular vesicles has clinical potential as a biomarker for disease. In biological samples, the smallest and largest vesicles typically differ 25-fold in size, 300,000-fold in concentration, 20,000-fold in volume, and 10,000,000-fold in scattered light. Because of this heterogeneity, the currently employed techniques detect concentrations ranging from 10(4) to 10(12) vesicles mL(-1) . OBJECTIVES: To investigate whether the large variation in the detected concentration of vesicles is caused by the minimum detectable vesicle size of five widely used techniques. METHODS: The size and concentration of vesicles and reference beads were measured with transmission electron microscopy (TEM), a conventional flow cytometer, a flow cytometer dedicated to detecting submicrometer particles, nanoparticle tracking analysis (NTA), and resistive pulse sensing (RPS). RESULTS: Each technique gave a different size distribution and a different concentration for the same vesicle sample. CONCLUSION: Differences between the detected vesicle concentrations are primarily caused by differences between the minimum detectable vesicle sizes. The minimum detectable vesicle sizes were 70-90 nm for NTA, 70-100 nm for RPS, 150-190 nm for dedicated flow cytometry, and 270-600 nm for conventional flow cytometry. TEM could detect the smallest vesicles present, albeit after adhesion on a surface. Dedicated flow cytometry was most accurate in determining the size of reference beads, but is expected to be less accurate on vesicles, owing to heterogeneity of the refractive index of vesicles. Nevertheless, dedicated flow cytometry is relatively fast and allows multiplex fluorescence detection, making it most applicable to clinical research.

Oral vaccination and protection of red foxes (Vulpes vulpes) against rabies using ONRAB, an adenovirus-rabies recombinant vaccine.

Twenty-seven red foxes (Vulpes vulpes) were each offered a bait containing ONRAB, a recombinant oral rabies vaccine that uses a human adenovirus vector to express the immunogenic rabies virus glycoprotein; 10 controls received no vaccine baits. Serum samples collected from all foxes before treatment, and each week post-treatment for 16 weeks, were tested for the presence of rabies virus neutralizing antibody (RVNA). In the bait group, a fox was considered a responder to vaccination if serum samples from 3 or more consecutive weeks had RVNA ≥0.5 IU/ml. Using this criterion, 79% of adult foxes (11/14) and 46% of juveniles (6/13) responded to vaccination with ONRAB. Serum RVNA of adults first tested positive (≥0.5 IU/ml) between weeks 1 and 3, about 4 weeks earlier than in juveniles. Adults also responded with higher levels of RVNA and these levels were maintained longer. Serum samples from juveniles tested positive for 1-4 consecutive weeks; in adults the range was 2-15 weeks, with almost half of adults maintaining titres above 0.5 IU/ml for 9 or more consecutive weeks. Based on the kinetics of the antibody response to ONRAB, the best time to sample sera of wild adult foxes for evidence of vaccination is 7-11 weeks following bait distribution. Thirty-four foxes (25 ONRAB, 9 controls) were challenged with vulpine street virus 547 days post-vaccination. All controls developed rabies whereas eight of 13 adult vaccinates (62%) and four of 12 juvenile vaccinates (33%) survived. All foxes classed as non-responders to vaccination developed rabies. Of foxes considered responders to vaccination, 80% of adults (8/10) and 67% of juveniles (4/6) survived challenge. The duration of immunity conferred to foxes would appear adequate for bi-annual and annual bait distribution schedules as vaccinates were challenged 1.5 years post-vaccination.

Microparticle association and heterogeneity of tumor-derived tissue factor in plasma: is it important for coagulation activation?

BACKGROUND: Tumor-derived tissue factor (TF) activates coagulation in vitro and in vivo in an orthotopic model of human pancreatic cancer. Here, we further characterized tumor-derived TF in this model. METHODS: Conditioned medium (CM) of L3.6pl human pancreatic tumor cells and plasma from nude mice bearing L3.6pl tumors were ultracentrifuged, and the pellets were filtered through membranes with different pore sizes. The size distribution of particles was analyzed in CM or plasma fractions with nanoparticle tracking and dynamic light scattering. Human TF antigen and activity were measured in pellets and supernatants with ELISA and clotting or thrombin generation assays, respectively. Human alternatively spliced TF (asTF) was measured with ELISA. Human TF and thrombin-antithrombin complex (TAT) concentrations were assessed in plasma of mice injected with filtered fractions of CM. RESULTS: Particles in both CM and plasma were < 0.4 µm. TF antigen and activity in the CM were mainly associated with microparticles (MP). Approximately 50% of antigen and 20% of activity were associated with particles of < 0.1 µm. Injection of < 0.1 µm particles into mice caused a 30% drop in platelet counts and an increase in TAT levels. In contrast, ~ 90% of TF antigen in tumor-bearing mice plasmas was non-sedimentable, whereas TF activity was exclusively associated with MP. Particles of < 0.1 µm and the supernatants of both CM and plasma gained TF activity after addition of exogenous phospholipids. Although asTF was found in MP-free CM supernatants, it was also present in CM and plasma pellets. CONCLUSIONS: Tumor-derived particles of < 0.1 µm and non-sedimentable TF are or can become procoagulant in the presence of phospholipids, and may contribute to the procoagulant potential of circulating TF.

An assessment of ONRAB oral rabies vaccine persistence in free-ranging mammal populations in Ontario, Canada.

ONRAB is a rabies glycoprotein recombinant human adenovirus type 5 oral vaccine developed for application in baits to control rabies in wildlife populations. Prior to widespread use of ONRAB, both the safety and effectiveness of this vaccine required investigation. While previous research has focused on field performance and the persistence and pathogenicity of ONRAB in captive animals, we sought to examine persistence and shedding of ONRAB in populations of free-ranging target and non-target mammals. We collected oral and rectal swab samples from 84 red foxes, 169 striped skunks, and 116 raccoons during 2007 and 2008 in areas where ONRAB vaccine baits were distributed. We also analyzed 930 tissue samples, 135 oral swab and 138 rectal swab samples from 155 non-target small mammals from 10 species captured during 2008 at sites treated with high densities of ONRAB vaccine baits. Samples were screened for the presence and quantity of ONRAB DNA using quantitative real-time PCR. None of the samples that we analyzed from target and non-target species contained quantities of ONRAB greater than 10(3)EU/mL of ONRAB DNA which is a limit that has previously been applied to assess viral shedding. This study builds on similar research and suggests that replication of ONRAB in animals is short-lived and the likelihood of horizontal transmission to other organisms is low.

Diagnosis of antiphospholipid syndrome in routine clinical practice.

The updated international consensus criteria for definite antiphospholipid syndrome (APS) are useful for scientific clinical studies. However, there remains a need for diagnostic criteria for routine clinical use. We audited the results of routine antiphospholipid antibodies (aPLs) in a cohort of 193 consecutive patients with aPL positivity-based testing for lupus anticoagulant (LA), IgG and IgM anticardiolipin (aCL) and anti-ß(2)glycoprotein-1 antibodies (aß(2)GPI). Medium/high-titre aCL/aß(2)GPI was defined as >99th percentile. Low-titre aCL/aß(2)GPI positivity (>95(th )< 99(th) percentile) was considered positive for obstetric but not for thrombotic APS. One hundred of the 145 patients fulfilled both clinical and laboratory criteria for definite APS. Twenty-six women with purely obstetric APS had persistent low-titre aCL and/or aß(2)GPI. With the inclusion of these patients, 126 of the 145 patients were considered to have APS. Sixty-seven out of 126 patients were LA-negative, of whom 12 had aCL only, 37 had aß(2)GPI only and 18 positive were for both. The omission of aCL or aß(2)GPI testing from investigation of APS would have led to a failure to diagnose APS in 9.5% and 29.4% of patients, respectively. Our data suggest that LA, aCL and aß(2)GPI testing are all required for the accurate diagnosis of APS and that low-titre antibodies should be included in the diagnosis of obstetric APS.