Pathology of Chronic Mycoplasma ovipneumoniae Carriers in a Declining Bighorn Sheep (Ovis canadensis) Population.

Bighorn sheep (Ovis canadensis) across North America commonly experience population-limiting epizootics of respiratory disease. Although many cases of bighorn sheep pneumonia are polymicrobial, Mycoplasma ovipneumoniae is most frequently associated with all-age mortality events followed by years of low recruitment. Chronic carriage of M. ovipneumoniae by adult females serves as a source of exposure of naïve juveniles; relatively few ewes may be responsible for maintenance of infection within a herd. Test-and-remove strategies focused on removal of adult females with evidence of persistent or intermittent shedding (hereafter chronic carriers) may reduce prevalence and mitigate mortality. Postmortem confirmation of pneumonia in chronic carriers has been inadequately reported and the pathology has not been thoroughly characterized, limiting our understanding of important processes shaping the epidemiology of pneumonia in bighorn sheep. Here we document postmortem findings and characterize the lesions of seven ewes removed from a declining bighorn sheep population in Wyoming, USA, following at least two antemortem detections of M. ovipneumoniae within a 14-mo period. We confirmed that 6/7 (85.7%) had variable degrees of chronic pneumonia. Mycoplasma ovipneumoniae was detected in the lung of 4/7 (57.1%) animals postmortem. Four (57.1%) had paranasal sinus masses, all of which were classified as inflammatory, hyperplastic lesions. Pasteurella multocida was detected in all seven (100%) animals, while Trueperella pyogenes was detected in 5/7 (71.4%). Our findings indicate that not all chronic carriers have pneumonia, nor do all have detectable M. ovipneumoniae in the lung. Further, paranasal sinus masses are a common but inconsistent finding, and whether sinus lesions predispose to persistence or result from chronic carriage remains unclear. Our findings indicate that disease is variable in chronic M. ovipneumoniae carriers, underscoring the need for further efforts to characterize pathologic processes and underlying mechanisms in this system to inform management.

Development and optimisation of a reception testing protocol designed to eliminate HCV in the UK prison population.

Background & Aims: Micro-elimination of hepatitis C virus (HCV) in high-risk populations is a feasible approach towards achieving the World Health Organization's targets for viral hepatitis elimination by 2030. Prisons represent an area of high HCV prevalence and so initiatives that improve testing and treatment of residents are needed to eliminate HCV from prisons. This initiative aimed to improve the HCV screening and treatment rates of new residents arriving at prisons in England. Methods: A rapid test and treat pathway was developed and implemented in 47 prisons in England between May 2019 and October 2021 as a healthcare service improvement initiative. Prison healthcare staff performed opt-out HCV testing for all new residents at each prison within 7 days of arrival, and those who were positive for HCV RNA were offered treatment with direct-acting antivirals (DAAs). The Hepatitis C Trust provided peer support for all residents on treatment and those who were released into the community. Results: Of 107,260 new arrivals, 98,882 (92.2%) were offered HCV antibody testing, 63,137 (63.9%) were tested and 1,848 were treated. Testing rates increased from 53.7% in Year 1 to 86.0% in Year 3. Between May 2020 and October 2021, 40,727 residents were tested, 2,286 residents were positive for HCV antibodies and 940 residents were HCV RNA positive, giving an antibody prevalence of 5.6% and an RNA prevalence of 2.3%. A total of 921 residents were referred for treatment and 915 initiated DAA treatment (97.3% of whom were HCV RNA positive). Conclusions: This initiative showed that an opt-out HCV test and treat initiative in prison receptions is feasible and can be adapted to the needs of individual prisons as a viable way to achieve HCV micro-elimination. Impact and implications: Prisons represent an area of high HCV prevalence and so initiatives that improve testing and treatment of residents are needed to eliminate HCV from prisons. The reception testing protocol improved HCV screening in new arrivals across 47 prisons in England and could be a viable way for countries to achieve HCV micro-elimination in their prison systems. The reception testing protocol presented here can be adapted to the individual needs of prisons, globally, to improve HCV screening and treatment in this setting.

Adrenocortical Responses to Daily Stressors Are Calibrated by Early Life Adversity: An Investigation of the Adaptive Calibration Model.

Studies examining the impact of early adversity on physiological responsivity to environmental challenges in later life yield a complex pattern of findings and ambiguity regarding the direction of effect, with some studies reporting heightened responses and others reporting dampened responses. One potential reason for these mixed findings is an oversimplified theoretical model surrounding the connection between early life stressor exposure and subsequent stress responsivity. The adaptive calibration model offersa contemporary set of assumptions aimed at providing a better understanding of the ways that early life experiences shape the stress response system to better align with current and future environments. The current study utilized a large subsample from the National Study of Daily Experiences (N = 1,605) to examine the extent to which the association between daily stressor exposure and cortisol levels varies across levels of early life adversity. Results revealed that those individuals who experienced extremely low levels of early life adversity displayed the greatest increase in cortisol levels across the day as daily stressor exposure increased. Alternatively, those individuals who experienced extremely high levels of early life adversity displayed almost no change in diurnal production of cortisol as daily stressor exposure increased. The results are discussed within the evolutionary-developmental context of the adaptive calibration model along with suggestions for future research.

Clinical Characteristics of Neurocysticercosis in a Peruvian Population-Based Epilepsy Cohort: A Descriptive Cross-Sectional Study of Baseline Clinical Intake.

(1) Background: This study presents the baseline characteristics of a community-level population of people with epilepsy (n = 1975) living in an area endemic for Taenia solium, the pathogen responsible for neurocysticercosis (NCC). (2) Methods: Participants were sequentially enrolled in a clinical cohort from 2007 to 2020 in Tumbes, Peru. All participants provided demographic and clinical history and received clinical evaluations. Diagnostics, including neuroimaging, cysticercosis serologies, and EEG, were obtained where possible. The data presented are from the cross-sectional baseline assessment of cohort participants. (3) Results: Approximately 38% of participants met the criteria for NCC. Those with NCC were more likely to have adult-onset epilepsy, as well as a longer duration of epilepsy, as compared to their counterparts without NCC. Overall, the data indicate a large treatment gap, with only approximately a quarter of the baseline population with prescriptions for anti-seizure medications. (4) Conclusions: These data reveal a high proportion of NCC among people living with epilepsy in these communities, with limited health care resources. At baseline, 74% of the population were not receiving anti-seizure treatments. Further analyses of these data will clarify the natural history of the disease for this population.

Development of small molecule inhibitors of natural killer group 2D receptor (NKG2D).

Natural killer group 2D (NKG2D) is a homodimeric activating immunoreceptor whose function is to detect and eliminate compromised cells upon binding to the NKG2D ligands (NKG2DL) major histocompatibility complex (MHC) molecules class I-related chain A (MICA) and B (MICB) and UL16 binding proteins (ULBP1-6). While typically present at low levels in healthy cells and tissue, NKG2DL expression can be induced by viral infection, cellular stress or transformation. Aberrant activity along the NKG2D/NKG2DL axis has been associated with autoimmune diseases due to the increased expression of NKG2D ligands in human disease tissue, making NKG2D inhibitors an attractive target for immunomodulation. Herein we describe the discovery and optimization of small molecule PPI (protein-protein interaction) inhibitors of NKG2D/NKG2DL. Rapid SAR was guided by structure-based drug design and accomplished by iterative singleton and parallel medicinal chemistry synthesis. These efforts resulted in the identification of several potent analogs (14, 21, 30, 45) with functional activity and improved LLE.

Sexually dimorphic extracellular vesicle responses after chronic spinal cord injury are associated with neuroinflammation and neurodegeneration in the aged brain.

BACKGROUND: Medical advances have made it increasingly possible for spinal cord injury (SCI) survivors to survive decades after the insult. But how SCI affects aging changes and aging impacts the injury process have received limited attention. Extracellular vesicles (EVs) are recognized as critical mediators of neuroinflammation after CNS injury, including at a distance from the lesion site. We have previously shown that SCI in young male mice leads to robust changes in plasma EV count and microRNA (miR) content. Here, our goal was to investigate the impact of biological sex and aging on EVs and brain after SCI. METHODS: Young adult age-matched male and female C57BL/6 mice were subjected to SCI. At 19 months post-injury, total plasma EVs were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis (NTA). EVs miR cargo was examined using the Fireplex® assay. The transcriptional changes in the brain were assessed by a NanoString nCounter Neuropathology panel and validated by Western blot (WB) and flow cytometry (FC). A battery of behavioral tests was performed for assessment of neurological function. RESULTS: Transcriptomic changes showed a high number of changes between sham and those with SCI. Sex-specific changes were found in transcription networks related to disease association, activated microglia, and vesicle trafficking. FC showed higher microglia and myeloid counts in the injured tissue of SCI/Female compared to their male counterparts, along with higher microglial production of ROS in both injured site and the brain. In the latter, increased levels of TNF and mitochondrial membrane potential were seen in microglia from SCI/Female. WB and NTA revealed that EV markers are elevated in the plasma of SCI/Male. Particle concentration in the cortex increased after injury, with SCI/Female showing higher counts than SCI/Male. EVs cargo analysis revealed changes in miR content related to injury and sex. Behavioral testing confirmed impairment of cognition and depression at chronic time points after SCI in both sexes, without significant differences between males and females. CONCLUSIONS: Our study is the first to show sexually dimorphic changes in brain after very long-term SCI and supports a potential sex-dependent EV-mediated mechanism that contributes to SCI-induced brain changes.

Clearance of Mycoplasma ovipneumoniae in Captive Bighorn Sheep (Ovis canadensis) Following Extended Oral Doxycycline Treatment.

Respiratory disease is a significant barrier for bighorn sheep (Ovis canadensis) conservation, and a need remains for management options in both captive and free-ranging populations. We treated Mycoplasma ovipneumoniae infection in six bighorn lambs and five bighorn yearlings at two captive research facilities with twice daily oral doxycycline for 8 wk or longer. Doses of 5 mg/kg twice daily mixed in formula for lambs and 10 mg/kg twice daily mixed in moistened pellets for older lambs and yearlings were tolerated well with minimal side effects. All animals in this case report remain Mycoplasma ovipneumoniae free over 2 yr later. Further evaluation is warranted to confirm efficacy of this therapeutic approach.

Using Opportunistic Samples to Monitor West Nile Virus Infection Status in Greater Sage-grouse (Centrocercus urophasianus) in Wyoming, USA (2020-22).

The frequency of arthropod-borne viral disease in naïve hosts is subject to change based on complex interactions among vector, host, virus, and external factors (e.g., climate). Thus, continual monitoring for both disease susceptibility and host infection dynamics is needed, especially for viruses that have proven detrimental to the health of wildlife hosts of conservation concern. The Greater Sage-grouse (Centrocercus urophasianus) is a gamebird of ecological and economic importance in the western United States for which population declines have led to a Near Threatened conservation status. Although these declines have mainly been attributed to habitat loss, West Nile virus (WNV) also poses a threat, with regional transmission potentially fueled by anthropogenic landscape alterations that may facilitate mosquito breeding. With limited WNV monitoring in Greater Sage-grouse after recognition of high susceptibility to mortality early after its initial detection in the western United States, the potential long-term impacts of WNV on this species are poorly understood. We used the plaque reduction neutralization test of filter paper strip-eluted sera to assess for anti-WNV antibodies, indicating prior infection, in opportunistically collected samples. From 2020 to 2022, 85 Greater Sage-grouse in Wyoming were sampled; none had anti-WNV antibodies. This result corroborates findings of previous studies documenting low seroprevalence. With the tenuous conservation status of the species, all potential population health risks should be considered in future management strategies, especially in the face of changing climate and landscapes.

Mortality in Wild Turkeys (Meleagris gallopavo) Associated with Natural Infection with H5N1 Highly Pathogenic Avian Influenza Virus (HPAIV) Subclade 2.3.4.4.

A Eurasian strain of H5N1 highly pathogenic avian influenza virus (HPAIV) was first detected in North America in December 2021 and has since been confirmed in numerous wild and domestic avian species. In April 2022, 41 Wild Turkeys (Meleagris gallopavo) were found dead in Johnson County, Wyoming, USA adjacent to a property with confirmed HPAIV in a backyard poultry flock. Oropharyngeal swabs were collected from 11 of the 41 turkeys and necropsy was performed on seven. Avian influenza virus RNA was detected in all 11 turkeys by real-time reverse-transcription PCR. Acute, multiorgan necrosis was observed grossly and identified in all seven turkeys evaluated by histopathology, most consistently in the lung, spleen, liver, gastrointestinal tract, and gonads. Lesions indicate high virulence of subclade 2.3.4.4b H5N1 HPAIV in Wild Turkeys, with infections presenting as clusters of acute mortality. Although documented cases of HPAIV in Wild Turkeys are rare, these findings signify a risk of spillback from domestic poultry, which may be heightened by the recent rise in backyard poultry ownership and the use of peridomestic habitat by wild birds. Additional research is needed to better understand the risk of disease transmission at the interface of Wild Turkeys and backyard poultry and the potential conservation and management implications of HPAIV in wild gallinaceous birds.

Abundance and diversity of Culicoides Latreille (Diptera: Ceratopogonidae) in southern Ontario, Canada.

BACKGROUND: Culicoides Latreille (Diptera: Ceratopogonidae) is a genus of hematophagous midges feeding on various vertebrate hosts and serving as a vector for numerous pathogens important to livestock and wildlife health. North American pathogens include bluetongue (BT) and epizootic hemorrhagic disease (EHD) viruses. Little is known about Culicoides spp. distribution and abundance and species composition in Ontario, Canada, despite bordering numerous U.S. states with documented Culicoides spp. and BT and EHD virus activity. We sought to characterize Culicoides spp. distribution and abundance and to investigate whether select meteorological and ecological risk factors influenced the abundance of Culicoides biguttatus, C. stellifer, and the subgenus Avaritia trapped throughout southern Ontario. METHODS: From June to October of 2017 to 2018, CDC-type LED light suction traps were placed on twelve livestock-associated sites across southern Ontario. Culicoides spp. collected were morphologically identified to the species level when possible. Associations were examined using negative binomial regression among C. biguttatus, C. stellifer, and subgenus Avaritia abundance, and select factors: ambient temperature, rainfall, primary livestock species, latitude, and habitat type. RESULTS: In total, 33,905 Culicoides spp. midges were collected, encompassing 14 species from seven subgenera and one species group. Culicoides sonorensis was collected from three sites during both years. Within Ontario, the northern trapping locations had a pattern of seasonal peak abundance in August (2017) and July (2018), and the southern locations had abundance peaks in June for both years. Culicoides biguttatus, C. stellifer, and subgenus Avaritia were significantly more abundant if ovine was the primary livestock species at trapping sites (compared to bovine). Culicoides stellifer and subgenus Avaritia were significantly more abundant at mid- to high-temperature ranges on trap days (i.e., 17.3-20.2 and 20.3-31.0 °C compared to 9.5-17.2 °C). Additionally, subgenus Avaritia were significantly more abundant if rainfall 4 weeks prior was between 2.7 and 20.1 mm compared to 0.0 mm and if rainfall 8 weeks prior was between 0.1 and 2.1 mm compared to 0.0 mm. CONCLUSIONS: Results from our study describe Culicoides spp. distribution in southern Ontario, the potential for spread and maintenance of EHD and BT viruses, and concurrent health risks to livestock and wildlife in southern Ontario in reference to certain meteorological and ecological risk factors. We identified that Culicoides spp. are diverse in this province, and appear to be distinctly distributed spatially and temporally. The livestock species present, temperature, and rainfall appear to have an impact on the abundance of C. biguttatus, C. stellifer, and subgenus Avaritia trapped. These findings could help inform targeted surveillance, control measures, and the development of management guides for Culicoides spp. and EHD and BT viruses in southern Ontario, Canada.

Identification of small-molecule protein-protein interaction inhibitors for NKG2D.

NKG2D (natural-killer group 2, member D) is a homodimeric transmembrane receptor that plays an important role in NK, γδ+, and CD8+ T cell-mediated immune responses to environmental stressors such as viral or bacterial infections and oxidative stress. However, aberrant NKG2D signaling has also been associated with chronic inflammatory and autoimmune diseases, and as such NKG2D is thought to be an attractive target for immune intervention. Here, we describe a comprehensive small-molecule hit identification strategy and two distinct series of protein-protein interaction inhibitors of NKG2D. Although the hits are chemically distinct, they share a unique allosteric mechanism of disrupting ligand binding by accessing a cryptic pocket and causing the two monomers of the NKG2D dimer to open apart and twist relative to one another. Leveraging a suite of biochemical and cell-based assays coupled with structure-based drug design, we established tractable structure-activity relationships with one of the chemical series and successfully improved both the potency and physicochemical properties. Together, we demonstrate that it is possible, albeit challenging, to disrupt the interaction between NKG2D and multiple protein ligands with a single molecule through allosteric modulation of the NKG2D receptor dimer/ligand interface.

Butorphanol, Azaperone, and Medetomidine for Chemical Immobilization in Free-Ranging Shiras (Alces alces shirasi) Moose: Ground and Helicopter Darting in Wyoming, USA.

We chemically immobilized a free-ranging moose subspecies, Shiras moose (Alces alces shirasi), using a combination of butorphanol (27.3 mg/mL), azaperone (9.1 mg/mL), and medetomidine (10.9 mg/mL). Ground and helicopter darting with fixed doses of 2 mL and 3 mL, respectively, safely immobilized 13 individuals in Wyoming, USA.

Utilizing blood filter paper and ear punch samples for the detection of rabbit hemorrhagic disease virus 2 by RT-rtPCR.

Rabbit hemorrhagic disease virus 2 (RHDV2), a virulent and contagious viral pathogen that affects wild and domestic lagomorph populations, was identified in Wyoming, USA in December 2020. A surveillance program was developed involving full-carcass submission and liver analysis, although carcass quality as a result of predation and decomposition impeded analysis. To increase the number of submissions and provide flexibility to field staff, we evaluated 2 sample types: 77 dried blood on filter paper samples, 66 ear punch samples. At initial sampling, test specificity and sensitivity of the RT-rtPCR utilizing dried blood on filter paper and ear punch samples were both 100% compared to liver. Filter paper results were consistent over time; sensitivity stayed >96% through weeks 2, 4, and 6, with a maximum mean difference of 6.0 Ct from baseline liver Ct values (95% CI: 5.0-7.3) at 6 wk. Test sensitivity of the ear punch sample at 1, 3, 5, and 7 wk post-sampling remained at 100%, with a maximum mean difference of 5.6 Ct from baseline liver Ct values (95% CI: 4.3-6.9) at 5 wk. Filter paper and ear punch samples were suitable alternatives to liver for RHDV2 surveillance in wild lagomorph populations. Alternative sampling options provide more flexibility to surveillance programs, increase testable submissions, and decrease exposure of field personnel to zoonotic disease agents.

Biopsychosocial sequelae of chronically painful injuries sustained in motor vehicle accidents contributing to non-recovery: A retrospective cohort study.

BACKGROUND: Claimants with chronically painful injuries sustained in motor vehicle accidents (MVAs) undergo assessment and management influenced by insurance and medico-legal processes defined by a biomedical paradigm which is discordant with best evidence. We aim to demonstrate the impact of biopsychosocial factors on post-MVA sequelae which contribute to non-recovery. METHODS: This was a retrospective cohort study of medico-legal documents and reports on 300 consecutive claimants referred to a pain medicine physician over 7 years (2012-2018) for assessment of painful musculoskeletal injuries post-MVA. One hundred data items were extracted from the medico-legal documents and reports for each claimant and entered into an electronic database. Post-MVA sequelae were analysed using chi-square analysis (OR >2) for significant associations with demographic, pre-MVA and post-MVA variables. Factors with significant associations were entered into a logistic regression model to determine significant statistical predictors of post-MVA sequelae contributing to non-recovery. RESULTS: The claimants were aged 17 to 80 years (mean age 42 years), and approximately half (53%, n=159) were female. The time from MVA to interview averaged 2.5 years. Widespread pain was present in 18% (n=54), and widespread somatosensory signs implying central sensitisation (OR=9.85, p<.001) was the most significant multivariate association. Long-term opiate use post-MVA (32%) was predicted by pre-MVA sleep disturbance (OR=5.08, p=.001), post-MVA major depressive disorder (MDD) (OR=3.02, p=.003) and long-term unemployment (OR=2.22. p=.007). Approximately half (47%, n=142) required post-MVA support from a psychologist or psychiatrist. Post-traumatic stress disorder (PTSD) was diagnosed by a psychiatrist or psychologist in 20% (n=59), yet early identification of risk of PTSD was rare. Pre-MVA, 89.4% (n=268) were studying or employed. Permanent unemployability post-MVA occurred in 35% (n=104) and was predicted by MDD (OR=3.59, p=.001) and antidepressant use (OR=2.17, p=.005). Major social change post-MVA (70%) was predicted by older age (OR=.966, p=.003), depressive symptoms (OR=3.71, p<.001) and opiate use (OR=2.00, p=.039). CONCLUSIONS: Biomedical factors, including older age, impaired sleep and indicators of widespread central sensitisation, and psychological factors, including stress, anxiety and depression, were the most prominent multivariate associations as statistical predictors of major adverse sequelae contributing to non-recovery for claimants with chronic pain post-MVA.

SOURCE AND SEASONALITY OF EPIZOOTIC MYCOPLASMOSIS IN FREE-RANGING PRONGHORN (ANTILOCAPRA AMERICANA).

Mycoplasma bovis is an economically important bacterial pathogen of cattle (Bos taurus) and bison (Bison bison) that most commonly causes pneumonia, polyarthritis, and mastitis. It is prevalent in cattle and ranched bison; however, infections in other species are rare. In early 2019, we identified M. bovis in free-ranging pronghorn (Antilocapra americana) in northeastern Wyoming. Here, we report on additional pronghorn mortalities caused by M. bovis, in the same approximately 120-km2 geographic region 1 yr later. Genetic analysis by multilocus sequence typing revealed that the mortalities were caused by the same M. bovis sequence type, which is unique among all sequence types documented thus far in North America. To explore whether pronghorn maintain chronic infections and begin assessing M. bovis status in other sympatric species, we used PCR testing of nasal swabs to opportunistically survey select free-ranging ungulates. We found no evidence of subclinical infections in 13 pronghorn sampled from the outbreak area (upper 95% binomial confidence limit [bCL], ∼24.7%) or among 217 additional pronghorn (upper 95% bCL, ∼1.7%) sampled from eight additional counties in Wyoming and 10 in Montana. All mule deer (Odocoileus hemionus; n=231; upper 95% bCL, ∼1.6%) sampled from 11 counties in Wyoming also were PCR negative. To assess the potential for environmental transmission, we examined persistence of M. bovis in various substrates and conditions. Controlled experiments revealed that M. bovis can remain viable for 6 h in shaded water and 2 h in direct sunlight. Our results indicate that environmental transmission of M. bovis from livestock to pronghorn is possible and that seasonality of infection could be due to shared resources during late winter. Further investigations to better understand transmission dynamics, to assess population level impacts to pronghorn, and to determine disease risks among pronghorn and other ungulate taxa appear warranted.

Functional and transcriptional profiling of microglial activation during the chronic phase of TBI identifies an age-related driver of poor outcome in old mice.

Elderly patients with traumatic brain injury (TBI) have greater mortality and poorer outcomes than younger individuals. The extent to which old age alters long-term recovery and chronic microglial activation after TBI is unknown, and evidence for therapeutic efficacy in aged mice is sorely lacking. The present study sought to identify potential inflammatory mechanisms underlying age-related outcomes late after TBI. Controlled cortical impact was used to induce moderate TBI in young and old male C57BL/6 mice. At 12 weeks post-injury, aged mice exhibited higher mortality, poorer functional outcomes, larger lesion volumes, and increased microglial activation. Transcriptomic analysis identified age- and TBI-specific gene changes consistent with a disease-associated microglial signature in the chronically injured brain, including those involved with complement, phagocytosis, and autophagy pathways. Dysregulation of phagocytic and autophagic function in microglia was accompanied by increased neuroinflammation in old mice. As proof-of-principle that these pathways have functional importance, we administered an autophagic enhancer, trehalose, in drinking water continuously for 8 weeks after TBI. Old mice treated with trehalose showed enhanced functional recovery and reduced microglial activation late after TBI compared to the sucrose control group. Our data indicate that microglia undergo chronic changes in autophagic regulation with both normal aging and TBI that are associated with poorer functional outcome. Enhancing autophagy may therefore be a promising clinical therapeutic strategy for TBI, especially in older patients.

Using whole-genome sequence data to examine the epidemiology of antimicrobial resistance in Escherichia coli from wild meso-mammals and environmental sources on swine farms, conservation areas, and the Grand River watershed in southern Ontario, Canada.

Antimicrobial resistance (AMR) threatens the health of humans and animals and has repeatedly been detected in wild animal species across the world. This cross-sectional study integrates whole-genome sequence data from Escherichia coli isolates with demonstrated phenotypic resistance that originated from a previous longitudinal wildlife study in southern Ontario, as well as phenotypically resistant E. coli water isolates previously collected as part of a public health surveillance program. The objective of this work was to assess for evidence of possible transmission of antimicrobial resistance determinants between wild meso-mammals, swine manure pits, and environmental sources on a broad scale in the Grand River watershed, and at a local scale-for the subset of samples collected on both swine farms and conservation areas in the previous wildlife study. Logistic regression models were used to assess potential associations between sampling source, location type (swine farm vs. conservation area), and the occurrence of select resistance genes and predicted plasmids. In total, 200 isolates from the following sources were included: water (n = 20), wildlife (n = 73), swine manure pit (n = 31), soil (n = 73), and dumpsters (n = 3). Several genes and plasmid incompatibility types were significantly more likely to be identified on swine farms compared to conservation areas. Conversely, internationally distributed sequence types (e.g., ST131), extended-spectrum beta-lactamase- and AmpC-producing E. coli were isolated in lower prevalences (<10%) and were almost exclusively identified in water sources, or in raccoon and soil isolates obtained from conservation areas. Differences in the odds of detecting resistance genes and predicted plasmids among various sources and location types suggest different primary sources for individual AMR determinants, but, broadly, our findings suggest that raccoons, skunks and opossums in this region may be exposed to AMR pollution via water and agricultural sources, as well as anthropogenic sources in conservation areas.

Rural Raccoons (Procyon lotor) Not Likely to Be a Major Driver of Antimicrobial Resistant Human Salmonella Cases in Southern Ontario, Canada: A One Health Epidemiological Assessment Using Whole-Genome Sequence Data.

Non-typhoidal Salmonella infections represent a substantial burden of illness in humans, and the increasing prevalence of antimicrobial resistance among these infections is a growing concern. Using a combination of Salmonella isolate short-read whole-genome sequence data from select human cases, raccoons, livestock and environmental sources, and an epidemiological framework, our objective was to determine if there was evidence for potential transmission of Salmonella and associated antimicrobial resistance determinants between these different sources in the Grand River watershed in Ontario, Canada. Logistic regression models were used to assess the potential associations between source type and the presence of select resistance genes and plasmid incompatibility types. A total of 608 isolates were obtained from the following sources: humans (n = 58), raccoons (n = 92), livestock (n = 329), and environmental samples (n = 129). Resistance genes of public health importance, including bla CMY-2, were identified in humans, livestock, and environmental sources, but not in raccoons. Most resistance genes analyzed were significantly more likely to be identified in livestock and/or human isolates than in raccoon isolates. Based on a 3,002-loci core genome multi-locus sequence typing (cgMLST) scheme, human Salmonella isolates were often more similar to isolates from livestock and environmental sources, than with those from raccoons. Rare instances of serovars S. Heidelberg and S. Enteritidis in raccoons likely represent incidental infections and highlight possible acquisition and dissemination of predominantly poultry-associated Salmonella by raccoons within these ecosystems. Raccoon-predominant serovars were either not identified among human isolates (S. Agona, S. Thompson) or differed by more than 350 cgMLST loci (S. Newport). Collectively, our findings suggest that the rural population of raccoons on swine farms in the Grand River watershed are unlikely to be major contributors to antimicrobial resistant human Salmonella cases in this region.

Using whole-genome sequence data to examine the epidemiology of Salmonella, Escherichia coli and associated antimicrobial resistance in raccoons (Procyon lotor), swine manure pits, and soil samples on swine farms in southern Ontario, Canada.

To better understand the contribution of wildlife to the dissemination of Salmonella and antimicrobial resistance in Salmonella and Escherichia coli, we examined whole-genome sequence data from Salmonella and E. coli isolates collected from raccoons (Procyon lotor) and environmental sources on farms in southern Ontario. All Salmonella and phenotypically resistant E. coli collected from raccoons, soil, and manure pits on five swine farms as part of a previous study were included. We assessed for evidence of potential transmission of these organisms between different sources and farms utilizing a combination of population structure assessments (using core-genome multi-locus sequence typing), direct comparisons of multi-drug resistant isolates, and epidemiological modeling of antimicrobial resistance (AMR) genes and plasmid incompatibility (Inc) types. Univariable logistic regression models were fit to assess the impact of source type, farm location, and sampling year on the occurrence of select resistance genes and Inc types. A total of 159 Salmonella and 96 resistant E. coli isolates were included. A diversity of Salmonella serovars and sequence types were identified, and, in some cases, we found similar or identical Salmonella isolates and resistance genes between raccoons, soil, and swine manure pits. Certain Inc types and resistance genes associated with source type were consistently more likely to be identified in isolates from raccoons than swine manure pits, suggesting that manure pits are not likely a primary source of those particular resistance determinants for raccoons. Overall, our data suggest that transmission of Salmonella and AMR determinants between raccoons and swine manure pits is uncommon, but soil-raccoon transmission appears to be occurring frequently. More comprehensive sampling of farms, and assessment of farms with other livestock species, as well as additional environmental sources (e.g., rivers) may help to further elucidate the movement of resistance genes between these various sources.

Discovery of an Allosteric, Inactive Conformation-Selective Inhibitor of Full-Length HPK1 Utilizing a Kinase Cascade Assay.

Achieving selectivity across the human kinome is a major hurdle in kinase inhibitor drug discovery. Assays using active, phosphorylated protein kinases bias hits toward poorly selective inhibitors that bind within the highly conserved adenosine triphosphate (ATP) pocket. Targeting inactive (vs active) kinase conformations offers advantages in achieving selectivity because of their more diversified structures. Kinase cascade assays are typically initiated with target kinases in their unphosphorylated inactive forms, which are activated during the assays. Therefore, these assays are capable of identifying inhibitors that preferentially bind to the unphosphorylated form of the enzyme in addition to those that bind to the active form. We applied this cascade assay to the emerging cancer immunotherapy target hematopoietic progenitor kinase 1 (HPK1), a serine/threonine kinase that negatively regulates T cell receptor signaling. Using this approach, we discovered an allosteric, inactive conformation-selective triazolopyrimidinone HPK1 inhibitor, compound 1. Compound 1 binds to unphosphorylated HPK1 >24-fold more potently than active HPK1, is not competitive with ATP, and is highly selective against kinases critical for T cell signaling. Furthermore, compound 1 does not bind to the isolated HPK1 kinase domain alone but requires other domains. Together, these data indicate that 1 is an allosteric HPK1 inhibitor that attenuates kinase autophosphorylation by binding to a pocket consisting of residues within and outside of the kinase domain. Our study demonstrates that cascade assays can lead to the discovery of highly selective kinase inhibitors. The triazolopyrimidinone described in this study may represent a privileged chemical scaffold for further development of potent and selective HPK1 inhibitors.