Induction of PrPSc-specific systemic and mucosal immune responses in white-tailed deer with an oral vaccine for chronic wasting disease

The ongoing epidemic of chronic wasting disease (CWD) within cervid populations indicates the need for novel approaches for disease management. A vaccine that either reduces susceptibility to infection or reduces shedding of prions by infected animals, or a combination of both, could be of benefit for disease control. The development of such a vaccine is challenged by the unique nature of prion diseases and the requirement for formulation and delivery in an oral format for application in wildlife settings. To address the unique nature of prions, our group targets epitopes, termed disease specific epitopes (DSEs), whose exposure for antibody binding depends on disease-associated misfolding of PrP^C into PrP^Sc. Here, a DSE corresponding to the rigid loop (RL) region, which was immunogenic following parenteral vaccination, was translated into an oral vaccine. This vaccine consists of a replication-incompetent human adenovirus expressing a truncated rabies glycoprotein G recombinant fusion with the RL epitope (hAd5:tgG-RL). Oral immunization of white-tailed deer with hAd5:tgG-RL induced PrP^Sc-specific systemic and mucosal antibody responses with an encouraging safety profile in terms of no adverse health effects nor prolonged vector shedding. By building upon proven strategies of formulation for wildlife vaccines, these efforts generate a particular PrP^Sc-specific oral vaccine for CWD as well as providing a versatile platform, in terms of carrier protein and biological vector, for generation of other oral, peptide-based CWD vaccines.     

Gene expression alterations in Rocky Mountain elk infected with chronic wasting disease

Chronic wasting disease (CWD) is an invariably fatal neurologic disease that naturally infects mule deer, white tailed deer and elk. The understanding of CWD neurodegeneration at a molecular level is very limited. In this study, microarray analysis was performed to determine changes in the gene expression profiles in six different tissues including brain, midbrain, thalamus, spleen, RPLN and tonsil of CWD-infected elk in comparison to non-infected healthy elk, using 24,000 bovine specific oligo probes. In total, 329 genes were found to be differentially expressed (> 2.0-fold) between CWD negative and positive brain tissues, with 132 genes upregulated and 197 genes downregulated. There were 249 DE genes in the spleen (168 up- and 81 downregulated), 30 DE genes in the retropharyngeal lymph node (RPLN) (18 up- and 12 downregulated), and 55 DE genes in the tonsil (21 up- and 34 downregulated). Using Gene Ontology (GO), the DE genes were assigned to functional groups associated with cellular process, biological regulation, metabolic process, and regulation of biological process. For all brain tissues, the highest ranking networks for DE genes identified by Ingenuity Pathway Analysis (IPA) were associated with neurological disease, cell morphology, cellular assembly and organization. Quantitative real-time PCR (qRT-PCR) validated the expression of DE genes primarily involved in different regulatory pathways, including neuronal signaling and synapse function, calcium signaling, apoptosis and cell death and immune cell trafficking and inflammatory response. This is the first study to evaluate altered gene expression in multiple organs including brain from orally infected elk and the results will improve our understanding of CWD neurodegeneration at the molecular level.     

Gene expression alterations in Rocky Mountain elk infected with chronic wasting disease

Chronic wasting disease (CWD) is an invariably fatal neurologic disease that naturally infects mule deer, white tailed deer and elk. The understanding of CWD neurodegeneration at a molecular level is very limited. In this study, microarray analysis was performed to determine changes in the gene expression profiles in six different tissues including brain, midbrain, thalamus, spleen, RPLN and tonsil of CWD-infected elk in comparison to non-infected healthy elk, using 24,000 bovine specific oligo probes. In total, 329 genes were found to be differentially expressed (> 2.0-fold) between CWD negative and positive brain tissues, with 132 genes upregulated and 197 genes downregulated. There were 249 DE genes in the spleen (168 up- and 81 downregulated), 30 DE genes in the retropharyngeal lymph node (RPLN) (18 up- and 12 downregulated), and 55 DE genes in the tonsil (21 up- and 34 downregulated). Using Gene Ontology (GO), the DE genes were assigned to functional groups associated with cellular process, biological regulation, metabolic process, and regulation of biological process. For all brain tissues, the highest ranking networks for DE genes identified by Ingenuity Pathway Analysis (IPA) were associated with neurological disease, cell morphology, cellular assembly and organization. Quantitative real-time PCR (qRT-PCR) validated the expression of DE genes primarily involved in different regulatory pathways, including neuronal signaling and synapse function, calcium signaling, apoptosis and cell death and immune cell trafficking and inflammatory response. This is the first study to evaluate altered gene expression in multiple organs including brain from orally infected elk and the results will improve our understanding of CWD neurodegeneration at the molecular level.     

同一健康理念下中国野生动物源疫病监测及响应体系发展对策

现代人类新发传染病中, 有60.3%是人兽共患病, 其中71.8%源于野生动物。野生动物是许多病原体的贮存库, 对人类和饲养动物会产生潜在的生物安全威胁。目前, 中国针对饲养动物疫病的监测检测系统和法律法规较为健全, 但针对野生动物的疫源疫病监测仍比较薄弱。根据“One Health”的理念, 野生动物疫源疫病的有效监测与相应防治措施的落实, 不仅可以为人兽共患病的大规模流行做出预警并降低其几率, 同时也为野生动物种群的健康提供了保障。本研究通过国际案例的比较分析, 提出有效的野生动物疫源疫病监测系统特征。同时, 通过对我国现有监测体系的研究分析, 结合利益相关方访谈以及实地调查, 提出了完善现有系统的主要措施建议。建议包括: (1)推进不同政府部门间的资源互通, 提高国家疫病监测体系应对跨学科、跨领域问题的综合能力; (2)针对人及饲养动物与野生动物接触频繁的生产生活方式, 应建立重点监测管理和响应机制; (3)提高对科学技术的重视, 包括建立野生动物疫病参考实验室、提升相关工作人员的技术能力等, 保障科学的监测方案和检测方法; (4)建立基于公众和现有监测资源的信息上报、汇总系统, 提升野生动物疫病监测的公众参与度和信息透明度。     

Applicability of current bovine spongiform encephalopathy (BSE) diagnostic procedures for chronic wasting disease (CWD)

Chronic wasting disease (CWD) in cervids is one of the transmissible spongiform encephalopathies ; however, its risk to humans is still obscure. An increase in number of diseased deer in North America has raised concerns regarding the CWD risk to humans. We demonstrated that the con-firmatory procedures and the commercial diagnostic kits for bovine spongiform encephalopathy (BSE) can be adopted for the diagnosis of CWD. No CWD case was confirmed in the surveillance of 558 cervids that were examined between 2003 and 2006 in Japan.     

Interaction among deer in a chronic wasting disease endemic zone

Although it is known that chronic wasting disease (CWD) can be transmitted by both direct animal-to-animal contact and contact with contaminated environments, the relative role of each mechanism in the spread of CWD in free-ranging populations has yet to be defined. We investigated patterns of interaction between mule deer (Odocoileus hemionus) in order to understand how factors such as season and landscape may influence patterns of disease spread in these populations. Using location data from male and female Global Positioning System (GPS)-collared mule deer in 5 study areas located in and around a CWD-endemic zone in southern Saskatchewan, Canada, we quantified close proximity events, or events involving both spatial and temporal overlap of individuals. We defined close proximity events as occurrences in which 2 deer were located <25 m apart at the same point in time. We looked at seasonal variation in the probability of close proximity events, as well as landscape factors associated with these events when compared to areas of shared space use, or spatial overlap alone. Overall probability of an individual GPS-collared deer being located in close proximity to another GPS-collared deer was 0.092 (n = 107). The early gestation (16 Dec–31 Mar) and late gestation (1 Apr–15 May) seasons had the highest probability of close proximity events occurring, and same-sex pairs were more likely to be found in close proximity than between-sex pairs during all seasons aside from the rut (1 Nov–15 Dec). High probability of close proximity events during the gestation seasons agrees with the tendency of mule deer to aggregate into large groups during late winter and suggests that this may be an important time period for CWD transmission to occur. Close proximity events occurred more in cropland and wetland than expected based on availability, whereas close proximity events occurred less than expected in grassland. The opposite was true for spatial overlap between individuals, which occurred more than expected in areas of low elevation and rugged terrain and in grassland or shrub–wood habitats. These results suggest that cropland may be a higher risk habitat for direct and indirect CWD transmission between individuals and that, although coulees and other areas of rugged topography are less likely to be associated with close proximity events, those areas may be more likely to contain environmental contamination in CWD-affected areas due to common use by multiple deer. © 2011 The Wildlife Society.     

Prion protein gene sequence and chronic wasting disease susceptibility in white-tailed deer (Odocoileus virginianus)

ABSTRACT

The sequence of the prion protein gene (PRNP) affects susceptibility to spongiform encephalopathies, or prion diseases in many species. In white-tailed deer, both coding and non-coding single nucleotide polymorphisms have been identified in this gene that correlate to chronic wasting disease (CWD) susceptibility. Previous studies examined individual nucleotide or amino acid mutations; here we examine all nucleotide polymorphisms and their combined effects on CWD. A 626 bp region of PRNP was examined from 703 free-ranging white-tailed deer. Deer were sampled between 2002 and 2010 by hunter harvest or government culling in Illinois and Wisconsin. Fourteen variable nucleotide positions were identified (4 new and 10 previously reported). We identified 68 diplotypes comprised of 24 predicted haplotypes, with the most common diplotype occurring in 123 individuals. Diplotypes that were found exclusively among positive or negative animals were rare, each occurring in less than 1% of the deer studied. Only one haplotype (C, odds ratio 0.240) and 2 diplotypes (AC and BC, odds ratios of 0.161 and 0.108 respectively) has significant associations with CWD resistance. Each contains mutations (one synonymous nucleotide 555C/T and one nonsynonymous nucleotide 286G/A) at positions reported to be significantly associated with reduced CWD susceptibility. Results suggest that deer populations with higher frequencies of haplotype C or diplotypes AC and BC might have a reduced risk for CWD infection – while populations with lower frequencies may have higher risk for infection. Understanding the genetic basis of CWD has improved our ability to assess herd susceptibility and direct management efforts within CWD infected areas.     

Legal regulation of supplementary cervid feeding facing chronic wasting disease

The supplementary feeding of cervids is a widespread practice across the northern hemisphere. There are few studies, however, regarding the extent of feeding in space and time. There are adverse effects of supplementary feeding, of which the most severe are increased parasite and disease transmission. With the recent emergence of chronic wasting disease (CWD) among cervids in Norway, a legal regulation was issued that banned all supplementary cervid feeding. We quantified the spatial extent and intentions of feeding cervids across all of Norway using a questionnaire at the municipality scale. We also compared spatial extent of feeding before and after the feeding ban to shed light on the ability of regulations to control supplementary feeding. Supplementary feeding to increase winter survival and targeting roe deer (Capreolus capreolus) was more common (48.4% of the municipalities) than the feeding of red deer (Cervus elaphus; 20.5%) and moose (Alces alces; 7.4%). The main feeding period was January–March, but extensive feeding also occurred from November to December and in April. Reducing traffic accidents was also a motivation, particularly for the feeding of moose (14.5%), and this was the main motivation (86%) for public feeding. Among the 65.7% that responded, 53.3% reported they knew about supplemental feeding of cervids in their municipality. In the region with the first feeding ban, 80.2% of municipalities were feeding in 2015–2016 before the ban, which was reduced to 68.4% in 2016–2017 and remained at 68.4% in 2017–2018. In the remainder of Norway, 81.4% were feeding in 2015–2016, and 72.6% were feeding in 2016–2017, but after the ban, this increased to 78.6% in the harsh winter of 2017–2018. Our study highlights that regulations across broad scales may not be followed and that more spatially targeted regulations and increased enforcement are required for disease transmission to be more effectively combated. © 2019 The Authors. Journal of Wildlife Management published by Wiley Periodicals, Inc. on behalf of The Wildlife Society.     

Species barriers for chronic wasting disease by in vitro conversion of prion protein

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy that can affect North American cervids (deer, elk, and moose). Using a novel in vitro conversion system based on incubation of prions with normal brain homogenates, we now report that PrP^CWD of elk can readily induce the conversion of normal cervid PrP (PrP^C) molecules to a protease-resistant form, but is less efficient in converting the PrP^C of other species, such as human, bovine, hamster, and mouse. However, when substrate brain homogenates are partially denatured by acidic conditions (pH 3.5), PrP^CWD-induced conversion can be greatly enhanced in all species. Our results demonstrate that PrP^C from cervids (including moose) can be efficiently converted to a protease-resistant form by incubation with elk CWD prions, presumably due to sequence and structural similarities between these species. Moreover, partial denaturation of substrate PrP^C can apparently overcome the structural barriers between more distant species.     

Tissue-specific biochemical differences between chronic wasting disease prions isolated from free-ranging white-tailed deer (Odocoileus virginianus)

Chronic wasting disease (CWD) is an invariably fatal prion disease affecting cervid species worldwide. Prions can manifest as distinct strains that can influence disease pathology and transmission. CWD is profoundly lymphotropic, and most infected cervids likely shed peripheral prions replicated in lymphoid organs. However, CWD is a neurodegenerative disease, and most research on prion strains has focused on neurogenic prions. Thus, a knowledge gap exists comparing neurogenic prions to lymphogenic prions. In this study, we compared prions from the obex and lymph nodes of naturally exposed white-tailed deer to identify potential biochemical strain differences. Here, we report biochemical evidence of strain differences between the brain and lymph node from these animals. Conformational stability assays, glycoform ratio analyses, and immunoreactivity scanning across the structured domain of the prion protein that refolds into the amyloid aggregate of the infectious prion reveal significantly more structural and glycoform variation in lymphogenic prions than neurogenic prions. Surprisingly, we observed greater biochemical differences among neurogenic prions than lymphogenic prions across individuals. We propose that the lymphoreticular system propagates a diverse array of prions from which the brain selects a more restricted pool of prions that may be quite different than those from another individual of the same species. Future work should examine the biological and zoonotic impact of these biochemical differences and examine more cervids from multiple locations to determine if these differences are conserved across species and locations.     

Influence of the geographic distribution of prion protein gene sequence variation on patterns of chronic wasting disease spread in white-tailed deer (Odocoileus virginianus)

ABSTRACT

Managing and controlling the spread of diseases in wild animal populations is challenging, especially for social and mobile species. Effective management benefits from information about disease susceptibility, allowing limited resources to be focused on areas or populations with a higher risk of infection. Chronic wasting disease (CWD), a transmissible spongiform encephalopathy that affects cervids, was detected in Colorado in the late 1960s. CWD was detected in Illinois and Wisconsin in 2002 and has since spread through many counties. Specific nucleotide variations in the prion protein gene (PRNP) sequence have been associated with reduced susceptibility to CWD in white-tailed deer. Though genetic resistance is incomplete, the frequency of deer possessing these mutations in a population is an important factor in disease spread (i.e. herd immunity). In this study we sequenced 625 bp of the PRNP gene from a sampling of 2433 deer from Illinois and Wisconsin. In north-central Illinois where CWD was first detected, counties had a low frequency of protective haplotypes (frequency <0.20); whereas in northwestern Illinois counties, where CWD cases have only more recently been detected, the frequency of protective haplotypes (frequency >0.30) was much higher (p < 0.05). Protective haplotype frequencies varied significantly among infected and uninfected geographic areas. The frequency of protective PRNP haplotypes may contribute to population level susceptibility and may shape the way CWD has spread through Illinois. Analysis of PRNP haplotype distribution could be a useful tool to assess CWD risk and allocate resources to contain and reduce the spread of infection.     

应用GIS技术进行野生动物生境研究概况及展望

1　 GIS的特性及对野生动物生境研究产生的影响近年来 ,生物多样性的研究和保护已在多层次、多水平上展开 [2 7] ,其中对野生动物生境的保护和研究是非常重要的一方面。生境 ( habitat)一词首先由美国的 Grinnel于 1 91 7年提出 ,指的是生物的居住场所 ,即生物个体、种群或群落能在其中完成生命过程的空间 [11]。自然界的生物都有它特定的生活环境 ,都有各自要求的适宜的环境条件。由于生物种类繁多 ,生物和环境、生物和生物之间的关系错综复杂 ,因此 ,野生动物生境的保护涉及到许多与时间动态及空间格局相关的问题。传统动物生态学和动物…     

Three-year follow-up of Interleukin 6 and C-reactive protein in chronic obstructive pulmonary disease

Background

Past studies have shown that mean values of Interleukin-6 (IL-6) and C-reactive protein (CRP) do not change significantly in COPD patients over a one-year period. However, longer period follow-up studies are still lacking. Thus, the aim of this study is to evaluate plasma CRP and IL-6 concentration over three years in COPD patients and to test the association between these inflammatory mediators and disease outcome markers.

Methods

A cohort of 77 outpatients with stable COPD was evaluated at baseline, and 53 (mean FEV₁, 56% predicted) were included in the prospective study. We evaluated Interleukin-6 (IL-6), C-reactive protein (CRP), six-minute walking distance (6MWD), and body mass index (BMI) at baseline and after three years. Plasma concentration of IL-6 was measured by high sensitivity ELISA, and CRP was obtained by high sensitivity particle-enhanced immunonephelometry.

Results

IL-6 increased significantly after 3 years compared to baseline measurements [0.8 (0.5-1.3) vs 2.4 (1.3-4.4) pg/ml; p < 0.001] and was associated with worse 6MWD performance. In the Cox regression, increased IL-6 at baseline was associated with mortality [Hazard Ratio (95% CI) = 2.68 (0.13, 1.84); p = 0.02]. CRP mean values did not change [5 (1.6-7.9) vs 4.7 (1.7-10) pg/L; p = 0.84], although eleven patients (21%) presented with changes >3 mg/L in CRP after 3 years.

Conclusions

The systemic inflammatory process, evaluated by IL-6, seems to be persistent, progressive and associated with mortality and worse physical performance in COPD patients.

Trial registration

No.:{"type":"clinical-trial","attrs":{"text":"NCT00605540","term_id":"NCT00605540"}}NCT00605540     

Commensal ecology, urban landscapes, and their influence on the genetic characteristics of city-dwelling Norway rats (Rattus norvegicus)

Movement of individuals promotes colonization of new areas, gene flow among local populations, and has implications for the spread of infectious agents and the control of pest species. Wild Norway rats ( Rattus norvegicus ) are common in highly urbanized areas but surprisingly little is known of their population structure. We sampled individuals from 11 locations within Baltimore, Maryland, to characterize the genetic structure and extent of gene flow between areas within the city. Clustering methods and a neighbour-joining tree based on pairwise genetic distances supported an east–west division in the inner city, and a third cluster comprised of historically more recent sites. Most individuals (~95%) were assigned to their area of capture, indicating strong site fidelity. Moreover, the axial dispersal distance of rats (62 m) fell within typical alley length. Several rats were assigned to areas 2–11.5 km away, indicating some, albeit infrequent, long-distance movement within the city. Although individual movement appears to be limited (30–150 m), locations up to 1.7 km are comprised of relatives. Moderate F _ST, differentiation between identified clusters, and high allelic diversity indicate that regular gene flow, either via recruitment or migration, has prevented isolation. Therefore, ecology of commensal rodents in urban areas and life-history characteristics of Norway rats likely counteract many expected effects of isolation or founder events. An understanding of levels of connectivity of rat populations inhabiting urban areas provides information about the spatial scale at which populations of rats may spread disease, invade new areas, or be eradicated from an existing area without reinvasion.     

Distinguishing epidemic waves from disease spillover in a wildlife population

Serengeti lions frequently experience viral outbreaks. In 1994, one-third of Serengeti lions died from canine distemper virus (CDV). Based on the limited epidemiological data available from this period, it has been unclear whether the 1994 outbreak was propagated by lion-to-lion transmission alone or involved multiple introductions from other sympatric carnivore species. More broadly, we do not know whether contacts between lions allow any pathogen with a relatively short infectious period to percolate through the population (i.e. reach epidemic proportions). We built one of the most realistic contact network models for a wildlife population to date, based on detailed behavioural and movement data from a long-term lion study population. The model allowed us to identify previously unrecognized biases in the sparse data from the 1994 outbreak and develop methods for judiciously inferring disease dynamics from typical wildlife samples. Our analysis of the model in light of the 1994 outbreak data strongly suggest that, although lions are sufficiently well connected to sustain epidemics of CDV-like diseases, the 1994 epidemic was fuelled by multiple spillovers from other carnivore species, such as jackals and hyenas.     

Assessing changes in clusters of wildlife road mortalities after the construction of wildlife mitigation structures

Collisions with vehicles can be a major threat to wildlife populations, so wildlife mitigation structures, including exclusionary fencing and wildlife crossings, are often constructed. To assess mitigation structure effectiveness, it is useful to compare wildlife road mortalities (WRMs) before, during, and after mitigation structure construction; however, differences in survey methodologies may make comparisons of counts impractical. Location‐based cluster analyses provide a means to assess how WRM spatial patterns have changed over time. We collected WRM data between 2015 and 2019 on State Highway 100 in Texas, USA. Five wildlife crossings and exclusionary fencing were installed in this area between September 2016 and May 2018 for the endangered ocelot (Leopardus pardalis) and other similarly sized mammals. Roads intersecting State Highway 100 were mitigated by gates, wildlife guards, and wing walls. However, these structures may have provided wildlife access to the highway. We combined local hot spot analysis and time series analysis to assess how WRM cluster intensity changed after mitigation structure construction at fine spatial and temporal scales and generalized linear regression to assess how gaps in fencing and land cover were related to WRM cluster intensity in the before, during, and after construction periods. Overall, WRMs/survey day decreased after mitigation structure construction and most hot spots occurred where there were more fence gaps, and, while cluster intensity increased in a few locations, these were not at fence gaps. Cluster intensity of WRMs increased when nearer to fence gaps in naturally vegetated areas, especially forested areas, and decreased nearer to fence gaps in areas with less natural vegetation. We recommend that if fence gaps are necessary in forested areas, less permeable mitigation structures, such as gates, should be used. Local hot spot analysis, coupled with time series and regression techniques, can effectively assess how WRM clustering changes over time.