Immunological and Pathogenic Differences of Two Experimental Bluetongue Virus Serotype Infections Evaluated in Two Disparate Host Species.

Bluetongue virus (BTV) is a prevalent midge-borne pathogen that infects ruminant species worldwide. BTV infections range from asymptomatic to lethal, with mechanisms that determine the severity of infection remaining largely undefined. Although it is relatively poorly understood, the immune response to BTV infection is thought to be critical for both the propagation of disease as well as the resolution of infection. To bridge this gap in knowledge, we infected cohorts of sheep and muntjac deer with two serotypes of BTV (BTV10 and BTV17) for longitudinal analysis (30 days). Interestingly, species-specific differences were observed. Circulating virus was detected early and remained detectable for the duration of the sheep study, while infections in muntjac showed faltering detection of BTV10 at 3 weeks post infection. The magnitude of the immune response was subdued in the muntjac when compared to the sheep cohorts, though similar responses were observed. We also assessed midge viral uptake and the ability to replicate BTV. Midges successfully fed on both species, yet those that fed on sheep resulted in more efficient BTV transmission. Our findings demonstrate that differences in BTV infections, immune responses, and vector competence across host species and serotypes will impact global BTV emergence and strategies for mitigation.

Evaluating Temperature Effects on Bluetongue Virus Serotype 10 and 17 Coinfection in Culicoides sonorensis.

Bluetongue virus (BTV) is a segmented, double-stranded RNA virus transmitted by Culicoides midges that infects ruminants. As global temperatures increase and geographical ranges of midges expand, there is increased potential for BTV outbreaks from incursions of novel serotypes into endemic regions. However, an understanding of the effect of temperature on reassortment is lacking. The objectives of this study were to compare how temperature affected Culicoides survival, virogenesis, and reassortment in Culicoides sonorensis coinfected with two BTV serotypes. Midges were fed blood meals containing BTV-10, BTV-17, or BTV serotype 10 and 17 and maintained at 20 °C, 25 °C, or 30 °C. Midge survival was assessed, and pools of midges were collected every other day to evaluate virogenesis of BTV via qRT-PCR. Additional pools of coinfected midges were collected for BTV plaque isolation. The genotypes of plaques were determined using next-generation sequencing. Warmer temperatures impacted traits related to vector competence in offsetting ways: BTV replicated faster in midges at warmer temperatures, but midges did not survive as long. Overall, plaques with BTV-17 genotype dominated, but BTV-10 was detected in some plaques, suggesting parental strain fitness may play a role in reassortment outcomes. Temperature adds an important dimension to host-pathogen interactions with implications for transmission and evolution.

Assessing Reassortment between Bluetongue Virus Serotypes 10 and 17 at Different Coinfection Ratios in Culicoides sonorenesis.

Bluetongue virus (BTV) is a segmented, double-stranded RNA orbivirus listed by the World Organization for Animal Health and transmitted by Culicoides biting midges. Segmented viruses can reassort, which facilitates rapid and important genotypic changes. Our study evaluated reassortment in Culicoides sonorensis midges coinfected with different ratios of BTV-10 and BTV-17. Midges were fed blood containing BTV-10, BTV-17, or a combination of both serotypes at 90:10, 75:25, 50:50, 25:75, or 10:90 ratios. Midges were collected every other day and tested for infection using pan BTV and cox1 (housekeeping gene) qRT-PCR. A curve was fit to the ∆Ct values (pan BTV Ct-cox1 Ct) for each experimental group. On day 10, the midges were processed for BTV plaque isolation. Genotypes of the plaques were determined by next-generation sequencing. Pairwise comparison of ∆Ct curves demonstrated no differences in viral RNA levels between coinfected treatment groups. Plaque genotyping indicated that most plaques fully aligned with one of the parental strains; however, reassortants were detected, and in the 75:25 pool, most plaques were reassortant. Reassortant prevalence may be maximized upon the occurrence of reassortant genotypes that can outcompete the parental genotypes. BTV reassortment and resulting biological consequences are important elements to understanding orbivirus emergence and evolution.

Integrating Veterinary Diagnostic Laboratories for Emergency Use Testing during Pandemics1.

The SARS-CoV-2 pandemic showed limitations in human outbreak testing. Veterinary diagnostic laboratories (VDLs) possess capabilities to bolster emergency test capacity. Surveys from 26 participating VDLs found human SARS-CoV-2 testing was mutually beneficial, including One Health benefits. VDLs indicated testing >3.8 million human samples during the pandemic, which included some challenges.

SARS-CoV-2 surveillance in a veterinary health system provides insight into transmission risks.

OBJECTIVE: To investigate the prevalence and seropositivity of SARS-CoV-2 in companion and exotic animals in a veterinary healthcare system. SAMPLE: A total of 341 animals were sampled by a combination of oral and nasal swabs. Serum from whole blood was collected from a subset of animals (86 canines, 25 felines, and 6 exotic animals). METHODS: After informed owner consent, convenience samples from client-owned animals and the pets of students and staff members associated with Colorado State University's Veterinary Health System were collected between May 2021 and September 2022. Study samples were collected by trained veterinarians, Veterinary Health System staff, and veterinary students. RESULTS: SARS-CoV-2 RNA was detected by reverse transcription PCR in 1.6% (95% CI, 0.5% to 4.6%) of domestic canines and 1.1% (95% CI, 0.2% to 6.1%) of domestic felines. No RNA was detected in any of the exotic animal species tested (n = 66). Plaque reduction neutralization tests indicated that 12.8% (95% CI, 7.3% to 21.5%) of canines and 12.0% (95% CI, 4.2% to 30.0%) of felines had neutralizing antibodies against SARS-CoV-2. CLINICAL RELEVANCE: This study provides insight regarding SARS-CoV-2 spillover in domestic companion and exotic animals and contributes to our understanding of transmission risk in the veterinary setting.

Recovery of multireassortant bluetongue virus serotype 6 sequences from a mule deer (Odocoileus hemionus) and Dorset sheep (Ovis aries) in Colorado.

We report the discovery of two bluetongue virus serotype 6 (BTV-6) reassortants recovered from a domestic sheep and a free-ranging mule deer in northern Colorado. At the time of this publication, whole-genome sequencing of BTV-6 isolates in the Western U.S. have not been undertaken. These findings reflect the incursive movement of geographically distinct BTV serotypes into important agricultural areas of the U.S. and demonstrate reassortment with regionally circulating serotypes.

Monitoring longitudinal immunological responses to bluetongue virus 17 in experimentally infected sheep.

Bluetongue virus (BTV) is an economically important pathogen of ruminant species with worldwide prevalence. While many BTV infections are asymptomatic, animals with symptomatic presentation deteriorate quickly with the sickest succumbing to disease within one week. Animals that survive the infection often require months to recover. The immune response to BTV infection is thought to play a central role in controlling the disease. Key to understanding BTV disease is profiling vertebrate host immunological cellular and cytokine responses. Studies to characterize immune responses in ruminants have been limited by a lack of species-specific reagents and assay technology. Here we assess the longitudinal immunological response to experimental BTV-17-California (CA) infection in sheep using the most up to date assays. We infected a cohort of sheep with BTV-17-CA and longitudinally monitored each animal for clinical disease, viremia and specific immunological parameters (B cells, T cells, monocytes) by RT-qPCR, traditional flow cytometry and/or fluorescent based antibody arrays. BTV-inoculated sheep exhibited clinical signs characteristic of bluetongue virus disease. Circulating virus was demonstrated after 8 days post inoculation (DPI) and remained detectable for the remainder of the time course (24 DPI). A distinct lymphopenia was observed between 7 and 14 DPI that rebounded to mock-inoculated control levels at 17 DPI. In addition, we observed increased expression of pro-inflammatory cytokines after 8 DPI. Taken together, we have established a model of BTV infection in sheep and have successfully monitored the longitudinal vertebrate host immunological response and viral infection progression using a combination of traditional methods and cutting-edge technology.

Culicoides-borne Orbivirus epidemiology in a changing climate.

Orbiviruses are of significant importance to the health of wildlife and domestic animals worldwide; the major orbiviruses transmitted by multiple biting midge (Culicoides) species include bluetongue virus, epizootic hemorrhagic disease virus, and African horse sickness virus. The viruses, insect vectors, and hosts are anticipated to be impacted by global climate change, altering established Orbivirus epidemiology. Changes in global climate have the potential to alter the vector competence and extrinsic incubation period of certain biting midge species, affect local and long-distance dispersal dynamics, lead to range expansion in the geographic distribution of vector species, and increase transmission period duration (earlier spring onset and later fall transmission). If transmission intensity is associated with weather anomalies such as droughts and wind speeds, there may be changes in the number of outbreaks and periods between outbreaks for some regions. Warmer temperatures and changing climates may impact the viral genome by facilitating reassortment and through the emergence of novel viral mutations. As the climate changes, Orbivirus epidemiology will be inextricably altered as has been seen with recent outbreaks of bluetongue, epizootic hemorrhagic disease, and African horse sickness outside of endemic areas, and requires interdisciplinary teams and approaches to assess and mitigate future outbreak threats.

In Situ Hybridization (RNAscope) Detection of Bluetongue Virus Serotypes 10 and 17 in Experimentally Co-Infected Culicoides sonorensis.

Bluetongue virus (BTV) is a segmented, double-stranded RNA virus transmitted by Culicoides biting midges. Infection of domestic and wild ruminants with BTV can result in a devastating disease and significant economic losses. As a virus with a segmented genome, reassortment among the BTV serotypes that have co-infected a host may increase genetic diversity, which can alter BTV transmission dynamics and generate epizootic events. The objective of this study was to determine the extent of dissemination and characterize the tropism of BTV serotypes 10 and 17 in co-infected Culicoides sonorensis. Midges were exposed to both BTV serotypes via blood meal and processed for histologic slides 10 days after infection. An in situ hybridization approach was employed using the RNAscope platform to detect the nucleic acid segment 2 of both serotypes. Observations of the mosaic patterns in which serotypes did not often overlap suggest that co-infection at the cellular level may not be abundant with these two serotypes in C. sonorensis. This could be a consequence of superinfection exclusion. Understanding BTV co-infection and its biological consequences will add an important dimension to the modeling of viral evolution and emergence.

Modeling cellular co-infection and reassortment of bluetongue virus in Culicoides midges.

When related segmented RNA viruses co-infect a single cell, viral reassortment can occur, potentially leading to new strains with pandemic potential. One virus capable of reassortment is bluetongue virus (BTV), which causes substantial health impacts in ruminants and is transmitted via Culicoides midges. Because midges can become co-infected by feeding on multiple different host species and remain infected for their entire life span, there is a high potential for reassortment to occur. Once a midge is co-infected, additional barriers must be crossed for a reassortant virus to emerge, such as cellular co-infection and dissemination of reassortant viruses to the salivary glands. We developed three mathematical models of within-midge BTV dynamics of increasing complexity, allowing us to explore the conditions leading to the emergence of reassortant viruses. In confronting the simplest model with published data, we estimate that the average life span of a bluetongue virion in the midge midgut is about 6 h, a key determinant of establishing a successful infection. Examination of the full model, which permits cellular co-infection and reassortment, shows that small differences in fitness of the two infecting strains can have a large impact on the frequency with which reassortant virions are observed. This is consistent with experimental co-infection studies with BTV strains with different relative fitnesses that did not produce reassortant progeny. Our models also highlight several gaps in existing data that would allow us to elucidate these dynamics in more detail, in particular the times it takes the virus to disseminate to different tissues, and measurements of viral load and reassortant frequency at different temperatures.

Bluetongue Research at a Crossroads: Modern Genomics Tools Can Pave the Way to New Insights.

Bluetongue virus (BTV) is an arthropod-borne, segmented double-stranded RNA virus that can cause severe disease in both wild and domestic ruminants. BTV evolves via several key mechanisms, including the accumulation of mutations over time and the reassortment of genome segments.Additionally, BTV must maintain fitness in two disparate hosts, the insect vector and the ruminant. The specific features of viral adaptation in each host that permit host-switching are poorly characterized. Limited field studies and experimental work have alluded to the presence of these phenomena at work, but our understanding of the factors that drive or constrain BTV's genetic diversification remains incomplete. Current research leveraging novel approaches and whole genome sequencing applications promises to improve our understanding of BTV's evolution, ultimately contributing to the development of better predictive models and management strategies to reduce future impacts of bluetongue epizootics.

Environmental Sustainability in Veterinary Medicine: An Opportunity for Teaching Hospitals.

Climate change is one of the greatest public health threats of the twenty-first century. Recent surveys of veterinary students and practicing veterinarians have highlighted their concerns about the impacts of climate change on animal health and a strong desire to be knowledgeable about the practice and promotion of environmental sustainability within clinical practice. Most American Veterinary Medical Association (AVMA)-accredited veterinary schools have a veterinary teaching hospital (VTH) where veterinary students receive their core clinical education. Given this, VTHs may provide opportunities for students to learn how veterinary clinics can decrease their environmental footprint and actions they could incorporate into their future clinical work. To assess the feasibility of and support for introducing environmentally sustainable practices into VTHs, we distributed an anonymous online survey to all AVMA-accredited veterinary schools with an associated VTH. Responses were received from 843 individuals representing 23 VTHs in 7 countries. While the overwhelming majority of responding personnel believe this is an important topic, there is little evidence that sustainable behaviors are being practiced or showcased within VTHs. Respondents were most interested in working to increase recycling and reduce general waste and energy consumption within their hospitals. In addition to a lack of educational resources, funding was a commonly identified barrier to incorporating more environmentally sustainable practices. These results add to the growing evidence that enhanced incorporation of sustainability into veterinary medical education at all stages is needed and that VTHs provide a unique opportunity to lead by example.

Exposure of Culicoides sonorensis to Enzootic Strains of Bluetongue Virus Demonstrates Temperature- and Virus-Specific Effects on Virogenesis.

Bluetongue virus (BTV) is a segmented RNA virus transmitted by Culicoides midges. Climatic factors, animal movement, vector species, and viral mutation and reassortment may all play a role in the occurrence of BTV outbreaks among susceptible ruminants. We used two enzootic strains of BTV (BTV-2 and BTV-10) to explore the potential for Culicoides sonorensis, a key North American vector, to be infected with these viruses, and identify the impact of temperature variations on virogenesis during infection. While BTV-10 replicated readily in C. sonorensis following an infectious blood meal, BTV-2 was less likely to result in productive infection at biologically relevant exposure levels. Moreover, when C. sonorensis were co-exposed to both viruses, we did not detect reassortment between the two viruses, despite previous in vitro findings indicating that BTV-2 and BTV-10 are able to reassort successfully. These results highlight that numerous factors, including vector species and exposure dose, may impact the in vivo replication of varying BTV strains, and underscore the complexities of BTV ecology in North America.

Preparing Veterinarians to Address the Health Impacts of Climate Change: Student Perceptions, Knowledge Gaps, and Opportunities.

Climate change poses a significant and increasing threat to global health. While veterinarians play integral roles in public and environmental health, the profession has been less engaged in the topic of climate change relative to their human medical counterparts. The objective of this study was to synthesize veterinary students' perceptions and knowledge about the relationship between veterinary medicine and climate change to identify educational gaps and opportunities. An online questionnaire was disseminated to American Veterinary Medical Association (AVMA)-accredited veterinary colleges through the Student American Veterinary Medical Association newsletter. Respondents were overwhelmingly confident that climate change is happening, is due to human activities, and is impacting both human and animal health. Veterinary students also expressed the belief that veterinarians should take a leadership role on the issue of climate change, especially through promoting environmental sustainability in clinical practice. Despite this enthusiasm, most students reported a lack of educational opportunities within their veterinary programs. The results of this survey highlight opportunities for the development of educational resources on the topic, many of which could complement existing material such as that devoted to practice management, economics, and client communication. Veterinarians have the potential to meaningfully contribute to both mitigation and adaptation efforts around climate change; however, they must be equipped with the tools to do so.

What Is Really Needed to Provide Effective, Person-Centered Care for Behavioral Expressions of Dementia? Guidance from The Alzheimer's Association Dementia Care Provider Roundtable.

In April, 2019, the Alzheimer's Association Dementia Care Provider Roundtable convened to discuss common challenges faced when implementing person-centered, non-pharmacological practices in long-term care and other settings that provide care and programs for persons living with dementia, and to develop relevant, specific guidance from the perspective of administrative leaders from 23 long-term and community-based care provider organizations (representing home, community-based, and residential care). Guidance related to 5 practice areas emerged from the facilitated discussion: having a foundational person-centered culture, conceptualizing behaviors as expressions and focusing on behavioral support, identifying antecedents and placing person-centeredness before protocols, modifying training to promote person-centered culture, and valuing implementation flexibility. In developing the practice guidance, a related list of priority areas for research and policy were also identified.

Ecological Dynamics Impacting Bluetongue Virus Transmission in North America.

Bluetongue virus (BTV) is an arbovirus transmitted to domestic and wild ruminants by certain species of Culicoides midges. The disease resulting from infection with BTV is economically important and can influence international trade and movement of livestock, the economics of livestock production, and animal welfare. Recent changes in the epidemiology of Culicoides-transmitted viruses, notably the emergence of exotic BTV genotypes in Europe, have demonstrated the devastating economic consequences of BTV epizootics and the complex nature of transmission across host-vector landscapes. Incursions of novel BTV serotypes into historically enzootic countries or regions, including the southeastern United States (US), Israel, Australia, and South America, have also occurred, suggesting diverse pathways for the transmission of these viruses. The abundance of BTV strains and multiple reassortant viruses circulating in Europe and the US in recent years demonstrates considerable genetic diversity of BTV strains and implies a history of reassortment events within the respective regions. While a great deal of emphasis is rightly placed on understanding the epidemiology and emergence of BTV beyond its natural ecosystem, the ecological contexts in which BTV maintains an enzootic cycle may also be of great significance. This review focuses on describing our current knowledge of ecological factors driving BTV transmission in North America. Information presented in this review can help inform future studies that may elucidate factors that are relevant to longstanding and emerging challenges associated with prevention of this disease.

Client Choice May Provide an Economic Incentive for Veterinary Practices to Invest in Sustainable Infrastructure and Climate Change Education.

Objective: To assess how pet owners perceive the role of veterinary medicine in addressing climate change and animal health and determine if there is a client-driven economic incentive to establish sustainable veterinary business practices. Sample: 1,044 dog and/or cat owners residing in the United States who had used veterinary services within the last 3 years. Procedures: An online Amazon mTurk survey about climate change and the perceived effects on client-owned dogs and cats was distributed to pet owners. Results: Most respondents believe climate change is occurring, and two-thirds of pet owners would value knowing their veterinarian received training on the animal health impacts of climate change. Over half of the respondents would pay more for veterinary services at a clinic with a reduced environmental impact. Additionally, clients would value some form of sustainability certification to aid in identification of such practices. Demographic influences found to be statistically significant included age, political ideology and where one resides (i.e., urban, suburban, or rural) whereas gender and income level, were not found to be significant. Conclusions and Clinical Relevance: Our data suggest there is an economic incentive for veterinary professionals to be knowledgeable about the health impacts of climate change and to implement and market sustainable practice initiatives. Prioritizing sustainable practice initiatives and climate change education in veterinary practices has the potential to mutually benefit both practitioner and client through shared patient health and financial incentives.

First trimester transabdominal chorionic villus sampling--does the needle matter?

PURPOSE: To compare first-trimester transabdominal chorionic villus samples (TA-CVS) when obtained by 20-gauge amniocentesis versus lancet needles. METHODS: This is a retrospective study of all women with viable singleton pregnancies undergoing TA-CVS from 01/01/2009 to 03/31/2011. All CVS were performed by a single operator using a freehand technique and amniocentesis needles from 01/01/2009 to 08/31/2010 and lancet needles from 09/01/2010 to 03/31/2011. All samples were processed by the same laboratory. RESULTS: There were no differences between groups regarding maternal age, weight, gestational age at CVS, indication for CVS, uterine position, or placental location. Lancet needles were associated with significantly larger samples (median 18 [range 3-40] versus 7 [range 1-33] mg, p < 0.0001), more successful in situ hybridization (96% versus 74.2%, p = 0.03), and faster result reporting (median 7 [range 5-12] versus 9 [range 6-26] days, p = 0.002). CONCLUSIONS: Needle type may be clinically important when selecting 20-gauge TA-CVS needles.

Stepwise sequential aneuploidy screening in clinical practice.

PURPOSE: To evaluate stepwise sequential screening (SSS) efficiency in clinical practice. METHODS: All singletons undergoing SSS in a single practice by NTQR (Nuchal Translucency Quality Review Program)-credentialed providers in a 2-year period were included. Prenatal diagnosis was offered to all screen-positive women and those with a nuchal translucency ≥3.5 mm or cystic hygroma at the 11- to 14-week scan. Data were extracted from prospectively ascertained serum screening and genetics databases. RESULTS: A total of 2,726 patients were screened, with SSS detecting all eight cases of trisomy 21 and all seven cases of other aneuploidies at a 4.3% screen-positive rate. CONCLUSIONS: Stepwise sequential screening offers excellent aneuploidy screening efficiency when introduced into clinical practice.