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.

Role of Spillover and Spillback in SARS-CoV-2 Transmission and the Importance of One Health in Understanding the Dynamics of the COVID-19 Pandemic.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is notable both for its impact on global public health as well as its well-publicized transmissibility to other species of animals. Infection of incidental animal hosts is concerning because of possible emergence of novel variants due to viral mutation. Species that are susceptible to SARS-CoV-2 include domestic and nondomestic cats, domestic dogs, white-tailed deer, mink, and golden hamsters, among others. We detail possible origins of transmission of SARS-CoV-2 to humans, as well as the ecological and molecular mechanisms needed for the virus to establish infection in humans from animals. We highlight examples of SARS-CoV-2 spillover, spillback, and secondary spillover, demonstrating the breadth in the variability of hosts and current transmission events that have been documented in domestic, captive, and wild animals. Lastly, we turn our focus to the importance of animal hosts as potential reservoirs and sources of variant emergence that can have profound effects on the human population. We note that a One Health approach emphasizing surveillance of animals and humans in certain environments using interdisciplinary collaboration is encouraged to manage disease surveillance, regulation on animal trade and testing, and animal vaccine development that will mitigate further disease outbreaks. These efforts will minimize the spread of SARS-CoV-2 and advance knowledge to prevent the spread of future emerging infectious diseases.

When dermatologic diseases are devastating: differentiating common endemic conditions in the United States from sheep and goat pox.

This article provides information to help US-based practitioners develop differential diagnoses for, and recognize foreign animal diseases associated with, dermatologic lesions in small ruminants. Sheep and goat pox are currently considered foreign animal diseases (in the United States) and may cause lesions similar to other endemic diseases of small ruminants including orf, ulcerative dermatosis, bluetongue, and dermatophilosis. Any cases involving unusual dermatologic lesions associated with high morbidity and/or mortality warrant reporting to governmental authorities including USDA APHIS or state regulatory veterinarians for herd or flock investigations. Vigilance on the part of livestock veterinarians and small ruminant producers is of paramount importance in preventing the entry and spread of economically devastating foreign animal diseases.

The Genetic Diversification of a Single Bluetongue Virus Strain Using an In Vitro Model of Alternating-Host Transmission.

Bluetongue virus (BTV) is an arbovirus that has been associated with dramatic epizootics in both wild and domestic ruminants in recent decades. As a segmented, double-stranded RNA virus, BTV can evolve via several mechanisms due to its genomic structure. However, the effect of BTV's alternating-host transmission cycle on the virus's genetic diversification remains poorly understood. Whole genome sequencing approaches offer a platform for investigating the effect of host-alternation across all ten segments of BTV's genome. To understand the role of alternating hosts in BTV's genetic diversification, a field isolate was passaged under three different conditions: (i) serial passages in Culicoides sonorensis cells, (ii) serial passages in bovine pulmonary artery endothelial cells, or (iii) alternating passages between insect and bovine cells. Aliquots of virus were sequenced, and single nucleotide variants were identified. Measures of viral population genetics were used to quantify the genetic diversification that occurred. Two consensus variants in segments 5 and 10 occurred in virus from all three conditions. While variants arose across all passages, measures of genetic diversity remained largely similar across cell culture conditions. Despite passage in a relaxed in vitro system, we found that this BTV isolate exhibited genetic stability across passages and conditions. Our findings underscore the valuable role that whole genome sequencing may play in improving understanding of viral evolution and highlight the genetic stability of BTV.

Maternal Influenza A Virus Infection Restricts Fetal and Placental Growth and Adversely Affects the Fetal Thymic Transcriptome.

Maternal influenza A viral infections in humans are associated with low birth weight, increased risk of pre-term birth, stillbirth and congenital defects. To examine the effect of maternal influenza virus infection on placental and fetal growth, pregnant C57BL/6 mice were inoculated intranasally with influenza A virus A/CA/07/2009 pandemic H1N1 or phosphate-buffered saline (PBS) at E3.5, E7.5 or E12.5, and the placentae and fetuses collected and weighed at E18.5. Fetal thymuses were pooled from each litter. Placentae were examined histologically, stained by immunohistochemistry (IHC) for CD34 (hematopoietic progenitor cell antigen) and vascular channels quantified. RNA from E7.5 and E12.5 placentae and E7.5 fetal thymuses was subjected to RNA sequencing and pathway analysis. Placental weights were decreased in litters inoculated with influenza at E3.5 and E7.5. Placentae from E7.5 and E12.5 inoculated litters exhibited decreased labyrinth development and the transmembrane protein 150A gene was upregulated in E7.5 placentae. Fetal weights were decreased in litters inoculated at E7.5 and E12.5 compared to controls. RNA sequencing of E7.5 thymuses indicated that 957 genes were downregulated ≥2-fold including Mal, which is associated with Toll-like receptor signaling and T cell differentiation. There were 28 upregulated genes. It is concluded that maternal influenza A virus infection impairs fetal thymic gene expression as well as restricting placental and fetal growth.

Seroprevalence of Leptospira spp. in Colorado equids and association with clinical disease.

Detection of Leptospira interrogans is difficult as a result of intermittent leptospiruria and brief leptospiremia. Hence, diagnosis relies heavily on serologic testing, the reference method of which is the microscopic agglutination test (MAT). In horses, clinical leptospirosis has been associated with abortion, recurrent uveitis, and sporadic cases of hepatic and renal disease. Little information exists on the seroprevalence of antibodies to L. interrogans in equids in the United States; past nationwide studies suggest that the seroprevalence in some areas is as high as 77% (reciprocal titer ≥ 100). We tested sera from 124 apparently healthy horses previously submitted for equine infectious anemia (EIA) serology using MAT for 6 serovars-Bratislava, Canicola, Grippotyphosa, Hardjo, Icterohaemorrhagiae, and Pomona. When using a reciprocal MAT titer cutoff of ≥ 100, 102 of 124 (82%) of the samples were positive for at least one serovar. Seropositivity was significantly associated with increasing age. Query of specimens from clinical cases submitted to the Colorado State University Veterinary Diagnostic Laboratory for MAT since 2010 indicated significantly greater seroprevalence (p = 0.015) of pathogenic serovar Pomona in clinical cases compared to sera submitted from healthy equids for routine EIA testing. Information from our diagnostic laboratory submission forms also suggests a correlation between uveitis or other ophthalmic problems and serovar Pomona.

Laboratory evaluation of stable isotope labeling of Culicoides (Diptera: Ceratopogonidae) for adult dispersal studies.

BACKGROUND: Stable isotope labeling is a promising method for use in insect mark-capture and dispersal studies. Culicoides biting midges, which transmit several important animal pathogens, including bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV), are small flies that develop in various semi-aquatic habitats. Previous Culicoides dispersal studies have suffered from the limitations of other labeling techniques, and an inability to definitively connect collected adult midges to specific immature development sites. RESULTS: Adult C. sonorensis were successfully labeled with 13C and 15N stable isotopes as larvae developing in a semi-aquatic mud substrate in the laboratory. High and low-dose isotope treatments for both elements significantly enriched midges above the background isotope levels of unenriched controls. Enrichment had no effect on C. sonorensis survival, though a slight (~ 5 day) delay in emergence was observed, and there was no significant effect of pool size on 13C or 15N enrichment levels. CONCLUSIONS: Stable isotope labeling is life-long, and does not interfere with natural insect behaviors. Stable isotope enrichment using 13C or 15N shows promise for Culicoides dispersal studies in the field. This method can be used to identify adult dispersal from larval source habitat where a midge developed. It may be possible to detect a single enriched midge in a pool of unenriched individuals, though further testing is needed to confirm the sensitivity of this method.

Bovine viral diarrhea in captive Rocky Mountain bighorn sheep associated with administration of a contaminated modified-live bluetongue virus vaccine.

In late summer 2017, we observed acute, fatal cases of bovine viral diarrhea in captive Rocky Mountain bighorn sheep ( Ovis canadensis canadensis) in Colorado following use of a contaminated modified-live bluetongue virus vaccine. Following vaccination, at least 14 of 28 (50%) vaccinated bighorn sheep developed hemorrhagic diarrhea, and 6 of 28 (21%) vaccinated bighorn sheep died. Autopsy findings were predominantly necroulcerative-to-necrohemorrhagic gastrointestinal lesions. Less frequent lesions included suffusive hemorrhages of serosal surfaces of abdominal viscera, and lymphoid necrosis in gut-associated lymphoid tissues. All of the 6 bighorn sheep that died were positive on real-time PCR (rtPCR) for bovine viral diarrhea virus (BVDV) in multiple tissues. Seroconversion to BVDV-1 and immunohistochemistry for BVDV in affected tissues confirmed rtPCR results. Next-generation sequencing confirmed a match between the infecting strain of BVDV-1b and the contaminated vaccine.

Low Temperature Tolerance of Culicoides sonorensis (Diptera: Ceratopogonidae) Eggs, Larvae, and Pupae From Temperate and Subtropical Climates.

Culicoides sonorensis Wirth and Jones biting midges are the primary North American vectors of bluetongue virus (BTV), which infects domestic ruminant livestock and can cause high morbidity and mortality. Both virus and vector exhibit highly seasonal activity patterns, even in subtropical climates like southern California. However, the exact mechanism of BTV and Culicoides overwintering has been debated. In this study, we examined the supercooling point (SCP) and lower lethal temperature of a laboratory colony of C. sonorensis eggs, larvae, and pupae, as well as of field-collected larvae and pupae from subtropical (California) and temperate (Colorado) climates. Larvae and pupae succumbed to temperatures higher than their respective SCPs, indicating death from prefreezing cold injury. Eggs were the most cold-tolerant life stage, and were able to tolerate 1-h exposures to temperatures as low as -20 °C without suffering complete mortality. Larvae were the least cold-tolerant life stage and suffered complete mortality at < -4 °C, while temperatures of -9 to -10 °C were required to kill all pupae. Larvae and embryos suffered chronic cold injury after exposure to subzero temperatures, which increased mortality. Field-collected larvae succumbed to slightly higher temperatures ( > -3 °C) than colony larvae, but did not differ significantly in their survival. Culicoides sonorensis immatures did not cold harden when exposed to 4 °C for 24 h before freezing. Results of this study indicate that C. sonorensis eggs are more tolerant of environmental stress than previously thought. Further work is needed on C. sonorensis winter survival in the field to corroborate laboratory studies.

Bluetongue virus infection creates light averse Culicoides vectors and serious errors in transmission risk estimates.

BACKGROUND: Pathogen manipulation of host behavior can greatly impact vector-borne disease transmission, but almost no attention has been paid to how it affects disease surveillance. Bluetongue virus (BTV), transmitted by Culicoides biting midges, is a serious disease of ruminant livestock that can cause high morbidity and mortality and significant economic losses. Worldwide, the majority of surveillance for Culicoides to assess BTV transmission risk is done using UV-light traps. Here we show that field infection rates of BTV are significantly lower in midge vectors collected using traps baited with UV light versus a host cue (CO2). METHODS: We collected Culicoides sonorensis midges in suction traps baited with CO2, UV-light, or CO2 + UV on three dairies in southern California to assess differences in the resulting estimated infection rates from these collections. Pools of midges were tested for BTV by qRT-PCR, and maximum likelihood estimates of infection rate were calculated by trap. Infection rate estimates were also calculated by trapping site within a dairy. Colonized C. sonorensis were orally infected with BTV, and infection of the structures of the compound eye was examined using structured illumination microscopy. RESULTS: UV traps failed entirely to detect virus both early and late in the transmission season, and underestimated virus prevalence by as much as 8.5-fold. CO2 + UV traps also had significantly lower infection rates than CO2-only traps, suggesting that light may repel infected vectors. We found very high virus levels in the eyes of infected midges, possibly causing altered vision or light perception. Collecting location also greatly impacts our perception of virus activity. CONCLUSIONS: Because the majority of global vector surveillance for bluetongue uses only light-trapping, transmission risk estimates based on these collections are likely severely understated. Where national surveillance programs exist, alternatives to light-trapping should be considered. More broadly, disseminated infections of many arboviruses include infections in vectors' eyes and nervous tissues, and this may be causing unanticipated behavioral effects. Field demonstrations of pathogen-induced changes in vector behavior are quite rare, but should be studied in more systems to accurately predict vector-borne disease transmission.

Seasonal and interseasonal dynamics of bluetongue virus infection of dairy cattle and Culicoides sonorensis midges in northern California--implications for virus overwintering in temperate zones.

Bluetongue virus (BTV) is the cause of an economically important arboviral disease of domestic and wild ruminants. The occurrence of BTV infection of livestock is distinctly seasonal in temperate regions of the world, thus we determined the dynamics of BTV infection (using BTV-specific real time reverse transcriptase polymerase chain reaction) among sentinel cattle and vector Culicoides sonorensis (C. sonorensis) midges on a dairy farm in northern California throughout both the seasonal and interseasonal (overwintering) periods of BTV activity from August 2012 until March 2014. The data confirmed widespread infection of both sentinel cattle and vector midges during the August-November period of seasonal BTV transmission, however BTV infection of parous female midges captured in traps set during daylight hours also was detected in February of both 2013 and 2014, during the interseasonal period. The finding of BTV-infected vector midges during mid-winter suggests that BTV may overwinter in northern California by infection of long-lived female C. sonorensis midges that were infected during the prior seasonal period of virus transmission, and reemerged sporadically during the overwintering period; however the data do not definitively preclude other potential mechanisms of BTV overwintering that are also discussed.

Whole genome sequencing and phylogenetic analysis of Bluetongue virus serotype 2 strains isolated in the Americas including a novel strain from the western United States.

Bluetongue is a potentially fatal arboviral disease of domestic and wild ruminants that is characterized by widespread edema and tissue necrosis. Bluetongue virus (BTV) serotypes 10, 11, 13, and 17 occur throughout much of the United States, whereas serotype 2 (BTV-2) was previously only detected in the southeastern United States. Since 1998, 10 other BTV serotypes have also been isolated from ruminants in the southeastern United States. In 2010, BTV-2 was identified in California for the first time, and preliminary sequence analysis indicated that the virus isolate was closely related to BTV strains circulating in the southeastern United States. In the current study, the whole genome sequence of the California strain of BTV-2 was compared with those of other BTV-2 strains in the Americas. The results of the analysis suggest co-circulation of genetically distinct viruses in the southeastern United States, and further suggest that the 2010 western isolate is closely related to southeastern strains of BTV. Although it remains uncertain as to how this novel virus was translocated to California, the findings of the current study underscore the need for ongoing surveillance of this economically important livestock disease.

Seasonal variation and impact of waste-water lagoons as larval habitat on the population dynamics of Culicoides sonorensis (Diptera:Ceratpogonidae) at two dairy farms in northern California.

The Sacramento (northern Central) Valley of California (CA) has a hot Mediterranean climate and a diverse ecological landscape that is impacted extensively by human activities, which include the intensive farming of crops and livestock. Waste-water ponds, marshes, and irrigated fields associated with these agricultural activities provide abundant larval habitats for C. sonorensis midges, in addition to those sites that exist in the natural environment. Within this region, C. sonorensis is an important vector of bluetongue (BTV) and related viruses that adversely affect the international trade and movement of livestock, the economics of livestock production, and animal welfare. To characterize the seasonal dynamics of immature and adult C. sonorensis populations, abundance was monitored intensively on two dairy farms in the Sacramento Valley from August 2012- to July 2013. Adults were sampled every two weeks for 52 weeks by trapping (CDC style traps without light and baited with dry-ice) along N-S and E-W transects on each farm. One farm had large operational waste-water lagoons, whereas the lagoon on the other farm was drained and remained dry during the study. Spring emergence and seasonal abundance of adult C. sonorensis on both farms coincided with rising vernal temperature. Paradoxically, the abundance of midges on the farm without a functioning waste-water lagoon was increased as compared to abundance on the farm with a waste-water lagoon system, indicating that this infrastructure may not serve as the sole, or even the primary larval habitat. Adult midges disappeared from both farms from late November until May; however, low numbers of parous female midges were detected in traps set during daylight in the inter-seasonal winter period. This latter finding is especially critical as it provides a potential mechanism for the "overwintering" of BTV in temperate regions such as northern CA. Precise documentation of temporal changes in the annual abundance and dispersal of Culicoides midges is essential for the creation of models to predict BTV infection of livestock and to develop sound abatement strategies.

Potential strategies for control of bluetongue, a globally emerging, Culicoides-transmitted viral disease of ruminant livestock and wildlife.

Bluetongue (BT) is a non-zoonotic arboviral disease of certain wild and domestic species of cloven-hoofed ungulates. The causative agent, bluetongue virus (BTV), is spread through temperate and tropical regions of the world by biting Culicoides midges. Control of BTV infection is complicated by the plurality of virus serotypes and the ubiquity and opportunistic feeding behavior of its midge vector. The global distribution of BTV infection has recently altered, perhaps driven in part by climatic influences on midge species resident in different regions. The goal of this review is to evaluate realistic strategies that might be utilized to control or prevent future outbreaks of BT and other Culicoides-transmitted diseases. Importantly, optimal control of emerging, rapidly evolving arbovirus diseases such as BT will require integrated countermeasures that mitigate all aspects of the virus's transmission cycle. This will best be accomplished using preventative, rather than purely reactive strategies.

The combination of abundance and infection rates of Culicoides sonorensis estimates risk of subsequent bluetongue virus infection of sentinel cattle on California dairy farms.

Bluetongue (BT) is an important viral disease of ruminants that is transmitted by hematophagous Culicoides midges. We examined the seasonal patterns of abundance and infection of Culicoides sonorensis at four dairy farms in the northern Central Valley of California to develop estimates of risk for bluetongue virus (BTV) transmission to cattle at each farm. These four farms were selected because of their similar meteorological conditions but varying levels of vector abundance and BTV infection of cattle. C. sonorensis midges were collected weekly at each farm during the seasonal transmission period, using three different trapping methods: traps baited with either carbon dioxide (CO(2)) alone or traps with CO(2) and UV light, and by direct aspiration of midges from sentinel cattle. Analysis of BTV-infected midges using group and serotype-specific quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR) assays confirmed that BTV serotypes 10, 11, 13 and 17 are all present in the region, but that midge infection rates and the number of BTV serotypes circulating differed markedly among the individual farms. Furthermore, more serotypes of BTV were present in midges than in sentinel cattle at individual farms where BTV circulated, and the virus was detected at each farm in midges prior to detection in cattle. BTV infection rates were remarkably lower among female C. sonorensis midges collected by CO(2) traps with UV light than among midges collected by either animal-baited aspirations or in CO(2) traps without light. A subsample of female midges examined from each collection method showed no overall differences in the proportion of female midges that had previously fed on a host. Findings from this study confirm the importance of using sensitive surveillance methods for both midge collection and virus detection in epidemiological studies of BTV infection, which is especially critical if the data are to be used for development of mathematical models to predict the occurrence of BTV infection of livestock.

Anthropogenic and meteorological factors influence vector abundance and prevalence of bluetongue virus infection of dairy cattle in California.

Bluetongue is an economically important arboviral disease of ruminants that is transmitted by hematophagous Culicoides midges. In light of dramatic recent changes in the global distribution of bluetongue virus (BTV), the goals of this study were to re-evaluate the prevalence of BTV infection of cattle and abundance of Culicoides midges on individual dairy farms in California. A serosurvey of adult dairy cattle confirmed that BTV infection is prevalent throughout much of the state, although the coastal northwestern region remains free of infection and prevalence varies markedly among farms in the remainder of the state. Intensive sampling for one year of 4 farms in the northern Central Valley of California showed that the abundance of Culicoides midges was markedly different and coincided with the prevalence of BTV infection of sentinel cattle on each farm. Mean maximum and minimum temperatures and other meteorological parameters were similar on all 4 farms, thus we speculate that particular management practices were responsible for both the increased midge abundance and prevalence of BTV infection of cattle at individual farms. Specifically, it is concluded that variation in vector abundance at individual farms most likely is the result of waste-water lagoon and irrigation management practices, leading to higher BTV infection rates among livestock held on farms with more waste-water lagoons and greater acreage of land for waste-water irrigation.

Bluetongue virus infection alters the impedance of monolayers of bovine endothelial cells as a result of cell death.

Bluetongue virus (BTV) is the cause of bluetongue, an emerging, arthropod-transmitted disease of ungulates. Bluetongue is characterized by vascular injury with hemorrhage, tissue infarction and widespread edema, lesions that are consistent with those of the so-called viral hemorrhagic fevers. To further investigate the pathogenesis of vascular injury in bluetongue, we utilized an electrical impedance assay and immunofluorescence staining to compare the effects of BTV infection on cultured bovine endothelial cells (bPAEC) with those of inducers of cell death (Triton X-100) and interendothelial gap formation (tissue necrosis factor [TNF]). The data confirm that the adherens junctions of BTV-infected bPAECs remained intact until 24h post-infection, and that loss of monolayer impedance precisely coincided with onset of virus-induced cell death. In contrast, recombinant bovine TNF-alpha caused rapid loss of bPAEC monolayer impedance that was associated with interendothelial gap formation and redistribution of VE-cadherin, but without early cell death. The data from these in vitro studies are consistent with a pathogenesis of bluetongue that involves virus-induced vascular injury leading to thrombosis, hemorrhage and tissue necrosis. However, the contribution of cytokine-induced interendothelial gap formation with subsequent edema and hypovolemic shock contributes to the pathogenesis of bluetongue remains to be fully characterized.