Genetic Characteristics of Salmonella Isolates Recovered From Reused Broiler Litter Over Three Successive Flocks.

Salmonella infections are a leading cause of bacterial food-borne illness worldwide. Infections are highly associated with the consumption of contaminated food, and in particular, chicken meat. The severity of Salmonella infections depends on the presence of antimicrobial resistance genes and virulence factors. While there are many studies which have investigated Salmonella strains isolated from postharvest chicken samples, there is a gap in our understanding of the genetic properties that influence the persistence of Salmonella in preharvest and in particular their makeup of antimicrobial resistance genes and virulence factors. We used whole genome sequencing and hierarchical clustering to characterize and classify the genetic diversity of Salmonella enterica isolates (n = 55) recovered from the litter of commercial broiler chicken raised in four colocated broiler houses of one integrated farm over three consecutive flocks. The chicken were raised under a newly adopted "No Antibiotics Ever" program, and copper sulfate was administered via drinking water. In-silico serovar prediction identified three S. enterica serovars: Enteritidis (n = 12), Kentucky (n = 40), and Senftenberg (n = 3). Antimicrobial susceptibility testing revealed that only one S. Kentucky isolate was resistant to streptomycin, while the remaining isolates were susceptible to all antibiotics tested. Metal resistance operons, including copper and silver, were identified chromosomally and on plasmids in serovar Senftenberg and Kentucky isolates, respectively, while serovar Enteritidis carried several virulence factors on plasmids. Serovar Kentucky isolates harboring metal resistance operons were the only Salmonella isolates recovered from the litter of third flock cohort. These results suggest that there might be environmental selection for Salmonella strains carrying plasmid-associated metal resistance and virulence genes, which could play a role in their persistence in litter.

An Aptamer-Based Proteomic Analysis of Plasma from Cats (Felis catus) with Clinical Feline Infectious Peritonitis.

Feline infectious peritonitis (FIP) is a systemic disease manifestation of feline coronavirus (FCoV) and is the most important cause of infectious disease-related deaths in domestic cats. FIP has a variable clinical manifestation but is most often characterized by widespread vasculitis with visceral involvement and/or neurological disease that is typically fatal in the absence of antiviral therapy. Using an aptamer-based proteomics assay, we analyzed the plasma protein profiles of cats who were naturally infected with FIP (n = 19) in comparison to the plasma protein profiles of cats who were clinically healthy and negative for FCoV (n = 17) and cats who were positive for the enteric form of FCoV (n = 9). We identified 442 proteins that were significantly differentiable; in total, 219 increased and 223 decreased in FIP plasma versus clinically healthy cat plasma. Pathway enrichment and associated analyses showed that differentiable proteins were related to immune system processes, including the innate immune response, cytokine signaling, and antigen presentation, as well as apoptosis and vascular integrity. The relevance of these findings is discussed in the context of previous studies. While these results have the potential to inform diagnostic, therapeutic, and preventative investigations, they represent only a first step, and will require further validation.

Lactobacillus acidophilus Expressing Murine Rotavirus VP8 and Mucosal Adjuvants Induce Virus-Specific Immune Responses.

Rotavirus diarrhea-associated illness remains a major cause of global death in children under five, attributable in part to discrepancies in vaccine performance between high- and low-middle-income countries. Next-generation probiotic vaccines could help bridge this efficacy gap. We developed a novel recombinant Lactobacillus acidophilus (rLA) vaccine expressing rotavirus antigens of the VP8* domain from the rotavirus EDIM VP4 capsid protein along with the adjuvants FimH and FliC. The upp-based counterselective gene-replacement system was used to chromosomally integrate FimH, VP8Pep (10 amino acid epitope), and VP8-1 (206 amino acid protein) into the L. acidophilus genome, with FliC expressed from a plasmid. VP8 antigen and adjuvant expression were confirmed by flow cytometry and Western blot. Rotavirus naïve adult BALB/cJ mice were orally immunized followed by murine rotavirus strain ECWT viral challenge. Antirotavirus serum IgG and antigen-specific antibody-secreting cell responses were detected in rLA-vaccinated mice. A day after the oral rotavirus challenge, fecal antigen shedding was significantly decreased in the rLA group. These results indicate that novel rLA constructs expressing VP8 can be successfully constructed and used to generate modest homotypic protection from rotavirus challenge in an adult murine model, indicating the potential for a probiotic next-generation vaccine construct against human rotavirus.

Are rabid raccoons (Procyon lotor) ready for the rapture? Determining the geographic origin of rabies virus-infected raccoons using RADcapture and microhaplotypes.

North America is recognized for the exceptional richness of rabies virus (RV) wildlife reservoir species. Management of RV is accomplished through vaccination targeting mesocarnivore reservoir populations, such as the raccoon (Procyon lotor) in Eastern North America. Raccoons are a common generalist species, and populations may reach high densities in developed areas, which can result in contact with humans and pets with potential exposures to the raccoon variant of RV throughout the eastern United States. Understanding the spatial movement of RV by raccoon populations is important for monitoring and refining strategies supporting the landscape-level control and local elimination of this lethal zoonosis. We developed a high-throughput genotyping panel for raccoons based on hundreds of microhaplotypes to identify population structure and genetic diversity relevant to rabies management programs. Throughout the eastern United States, we identified hierarchical population genetic structure with clusters that were connected through isolation-by-distance. We also illustrate that this genotyping approach can be used to support real-time management priorities by identifying the geographic origin of a rabid raccoon that was collected in an area of the United States that had been raccoon RV-free for 8 years. The results from this study and the utility of the microhaplotype panel and genotyping method will provide managers with information on raccoon ecology that can be incorporated into future management decisions.

Virulence factors and antimicrobial resistance profiles of Campylobacter isolates recovered from consecutively reused broiler litter.

IMPORTANCE: Campylobacter is a leading cause of foodborne illness in the United States due to consumption of contaminated or mishandled food products, often associated with chicken meat. Campylobacter is common in the microbiota of avian and mammalian gut; however, acquisition of antimicrobial resistance genes (ARGs) and virulence factors (VFs) may result in strains that pose significant threat to public health. Although there are studies investigating the genetic diversity of Campylobacter strains isolated from post-harvest chicken samples, there are limited data on the genome characteristics of isolates recovered from preharvest broiler production. Here, we show that Campylobacter jejuni and Campylobacter coli differ in their carriage of antimicrobial resistance and virulence factors may also differ in their ability to persist in litter during consecutive grow-out of broiler flocks. We found that presence/absence of virulence factors needed for evasion of host defense mechanisms and gut colonization played an integral role in differentiating Campylobacter strains.

Population genomic analysis of an emerging pathogen Lonsdalea quercina affecting various species of oaks in western North America.

Understanding processes leading to disease emergence is important for effective disease management and prevention of future epidemics. Utilizing whole genome sequencing, we studied the phylogenetic relationship and diversity of two populations of the bacterial oak pathogen Lonsdalea quercina from western North America (Colorado and California) and compared these populations to other Lonsdalea species found worldwide. Phylogenetic analysis separated Colorado and California populations into two Lonsdalea clades, with genetic divergence near species boundaries, suggesting long isolation and populations that differ in genetic structure and distribution and possibly their polyphyletic origin. Genotypes collected from different host species and habitats were randomly distributed within the California cluster. Most Colorado isolates from introduced planted trees, however, were distinct from three isolates collected from a natural stand of Colorado native Quercus gambelii, indicating cryptic population structure. The California identical core genotypes distribution varied, while Colorado identical core genotypes were always collected from neighboring trees. Despite its recent emergence, the Colorado population had higher nucleotide diversity, possibly due to its long presence in Colorado or due to migrants moving with nursery stock. Overall, results suggest independent pathogen emergence in two states likely driven by changes in host-microbe interactions due to ecosystems changes. Further studies are warranted to understand evolutionary relationships among L. quercina from different areas, including the red oak native habitat in northeastern USA.

Loss of West Nile virus genetic diversity during mosquito infection due to species-dependent population bottlenecks.

Vector competence (VC) refers to the efficiency of pathogen transmission by vectors. Each step in the infection of a mosquito vector constitutes a barrier to transmission that may impose bottlenecks on virus populations. West Nile virus (WNV) is maintained by multiple mosquito species with varying VC. However, the extent to which bottlenecks and VC are linked is poorly understood. Similarly, quantitative analyses of mosquito-imposed bottlenecks on virus populations are limited. We used molecularly barcoded WNV to quantify tissue-associated population bottlenecks in three variably competent WNV vectors. Our results confirm strong population bottlenecks during mosquito infection that are capable of dramatically reshaping virus population structure in a non-selective manner. In addition, we found that mosquitoes with differing VC uniquely shape WNV population structure: highly competent vectors are more likely to contribute to the maintenance of rare viral genotypes. These findings have important implications for arbovirus emergence and evolution.

Biodiversity indices and Random Forests reveal the potential for striped skunk (Mephitis mephitis) fecal microbial communities to function as a biomarker for oral rabies vaccination.

Wildlife disease surveillance and monitoring poses unique challenges when assessing rates of population vaccination, immunity, or infection prevalence. Non-invasively detected biomarkers can help reduce risk to both animal and field personnel during wildlife disease management activities. In this study, we investigated the utility of fecal microbiome data collected from captive striped skunks (Mephitis mephitis) in predicting rabies virus vaccination and infection status. We sequenced the hypervariable region 4 (V4) of the bacterial 16S gene and estimated alpha and beta diversity across timepoints in three groups of skunks: vaccination then rabies virus infection, sham vaccination then rabies virus infection, and rabies virus infected without vaccination. Alpha diversity did not differ among treatment groups but beta diversity between treatments was statistically significant. The phyla Firmicutes and Proteobacteria were dominant among all samples. Using Random Forests, we identified operational taxonomic units (OTUs) that greatly influenced classification of fecal samples into treatment groups. Each of these OTUs was correlated with fecal volatile organic compounds detected from the samples for companion treatment groups in another study. This research is the first to highlight striped skunk microbiome biodiversity as a vaccination biomarker which pushes the frontier on alternative methods for surveillance and monitoring of vaccination and disease in wildlife populations.

Temporal dynamics of the cecal and litter microbiome of chickens raised in two separate broiler houses.

In this study, we investigated the dynamics of the ceca and litter microbiome of chickens from post-hatch through pre-harvest. To achieve this, six hundred one-day old Cobb 500 broiler chicks were raised on floor pens for 49 days in two separate houses. We performed short-read and full-length sequencing of the bacterial 16S rRNA gene present in the meconium and in cecal and litter samples collected over the duration of the study. In addition, we determined the antimicrobial resistance (AMR) phenotype of Escherichia coli and Enterococcus spp. isolated from the meconium and the ceca of 49-day old chickens. We monitored the relative humidity, temperature, and ammonia in each house daily and the pH and moisture of litter samples weekly. The overall microbial community structure of the ceca and litter consistently changed throughout the course of the grow-out and correlated with some of the environmental parameters measured (p < 0.05). We found that the ceca and litter microbiome were similar in the two houses at the beginning of the experiment, but over time, the microbial community separated and differed between the houses. When we compared the environmental parameters in the two houses, we found no significant differences in the first half of the growth cycle (day 0-21), but morning temperature, morning humidity, and ammonia significantly differed (p < 0.05) between the two houses from day 22-49. Lastly, the prevalence of AMR in cecal E. coli isolates differed from meconium isolates (p < 0.001), while the AMR phenotype of cecal Enterococcus isolates differed between houses (p < 0.05).

Broiler house environment and litter management practices impose selective pressures on antimicrobial resistance genes and virulence factors of Campylobacter.

Campylobacter infections are a leading cause of bacterial diarrhea in humans globally. Infections are due to consumption of contaminated food products and are highly associated with chicken meat, with chickens being an important reservoir for Campylobacter. Here, we characterized the genetic diversity of Campylobacter species detected in broiler chicken litter over three consecutive flocks and determined their antimicrobial resistance and virulence factor profiles. Antimicrobial susceptibility testing and whole genome sequencing were performed on Campylobacter jejuni (n = 39) and Campylobacter coli (n = 5) isolates. All C. jejuni isolates were susceptible to all antibiotics tested while C. coli (n =4) were resistant to only tetracycline and harbored the tetracycline-resistant ribosomal protection protein (TetO). Virulence factors differed within and across grow houses but were explained by the isolates' flock cohort, species and multilocus sequence type. Virulence factors involved in the ability to invade and colonize host tissues and evade host defenses were absent from flock cohort 3 C. jejuni isolates as compared to flock 1 and 2 isolates. Our results show that virulence factors and antimicrobial resistance genes differed by the isolates' multilocus sequence type and by the flock cohort they were present in. These data suggest that the house environment and litter management practices performed imposed selective pressures on antimicrobial resistance genes and virulence factors. In particular, the absence of key virulence factors within the final flock cohort 3 isolates suggests litter reuse selected for Campylobacter strains that are less likely to colonize the chicken host.

Chicken Production and Human Clinical Escherichia coli Isolates Differ in Their Carriage of Antimicrobial Resistance and Virulence Factors.

Contamination of food animal products by Escherichia coli is a leading cause of foodborne disease outbreaks, hospitalizations, and deaths in humans. Chicken is the most consumed meat both in the United States and across the globe according to the U.S. Department of Agriculture. Although E. coli is a ubiquitous commensal bacterium of the guts of humans and animals, its ability to acquire antimicrobial resistance (AMR) genes and virulence factors (VFs) can lead to the emergence of pathogenic strains that are resistant to critically important antibiotics. Thus, it is important to identify the genetic factors that contribute to the virulence and AMR of E. coli. In this study, we performed in-depth genomic evaluation of AMR genes and VFs of E. coli genomes available through the National Antimicrobial Resistance Monitoring System GenomeTrackr database. Our objective was to determine the genetic relatedness of chicken production isolates and human clinical isolates. To achieve this aim, we first developed a massively parallel analytical pipeline (Reads2Resistome) to accurately characterize the resistome of each E. coli genome, including the AMR genes and VFs harbored. We used random forests and hierarchical clustering to show that AMR genes and VFs are sufficient to classify isolates into different pathogenic phylogroups and host origin. We found that the presence of key type III secretion system and AMR genes differentiated human clinical isolates from chicken production isolates. These results further improve our understanding of the interconnected role AMR genes and VFs play in shaping the evolution of pathogenic E. coli strains. IMPORTANCE Pathogenic Escherichia coli causes disease in both humans and food-producing animals. E. coli pathogenesis is dependent on a repertoire of virulence factors and antimicrobial resistance genes. Food-borne outbreaks are highly associated with the consumption of undercooked and contaminated food products. This association highlights the need to understand the genetic factors that make E. coli virulent and pathogenic in humans and poultry. This research shows that E. coli isolates originating from human clinical settings and chicken production harbor different antimicrobial resistance genes and virulence factors that can be used to classify them into phylogroups and host origins. In addition, to aid in the repeatability and reproducibility of the results presented in this study, we have made a public repository of the Reads2Resistome pipeline and have provided the accession numbers associated with the E. coli genomes analyzed.

Management and environmental factors influence the prevalence and abundance of food-borne pathogens and commensal bacteria in peanut hull-based broiler litter.

In this study, we conducted a longitudinal sampling of peanut hull-based litter from a farm under a "no antibiotics ever" program. Our objective was to determine broiler management practices and environmental factors that are associated with the occurrence of food-borne pathogens (Salmonella and Campylobacter) and the abundance of commensal bacteria (Escherichia coli, Enterococcus spp., and Staphylococcus spp.). Litter (n = 288) was collected from 4 broiler houses over three consecutive flocks, starting with a complete house cleanout and fresh peanut hull. Litter was sampled at the beginning of each grow-out cycle and at the end of the cycle. Logistic and linear regression models were used to model the relationships between pathogen prevalence, commensal abundance and management practices, and environmental factors. The number of flocks raised on litter, grow-out period, broiler house, litter pH, litter moisture, and house temperature were associated with the prevalence of pathogens and the abundance of commensal bacteria in litter. The final logistic model for pathogens showed that a higher probability of detecting Salmonella in litter was associated with the number of flocks raised on litter and the grow-out period. A higher probability of detecting Campylobacter in litter was associated with the number of flocks raised on litter, broiler house and the sections of the house, and the pH of litter. Our results suggest that management practices and environmental factors affect Salmonella and Campylobacter differently and suggest that each pathogen will require its own tailored intervention to stop their persistence in broiler litter.

Demographic and histopathologic features of dogs with abnormally high concentrations of hepatic copper.

BACKGROUND: Copper associated hepatopathy (CAH) has become an important and prevalent disease since the 1990's, coincidental with changes in copper (Cu) content in commercial dog foods. Knowing the demographic and histopathologic features related to hepatic Cu concentrations might aid in diagnosing CAH in dogs. HYPOTHESIS/OBJECTIVES: The primary aim was to identify demographic and histopathologic features associated with abnormally high hepatic Cu concentrations. ANIMALS: Dogs that underwent liver histopathology and Cu quantification at a veterinary diagnostic laboratory between July 2010 and February 2020. METHODS: Data was retrospectively collected from an electronic database. A Gaussian multiple regression model on the log scale was used to evaluate associations between hepatic Cu and a set of demographic and histologic features selected with machine learning methods. RESULTS: Of 4559 cases meeting criteria, 50% had hepatic Cu > 400 and 19% had Cu > 1000 ppm (parts per million) dry weight (reference range 120-400). Median hepatic Cu was 391 ppm, range 4.5 to 31500. Age was negatively associated (P < .02), but specific breeds (Doberman pinscher, Labrador retriever, and West Highland white terrier) were positively associated with abnormally high hepatic Cu (P < .001). Severity of inflammation (mild, moderate, and severe) and necrosis/apoptosis were associated with abnormally high hepatic Cu (P < .01). CONCLUSION AND CLINICAL IMPORTANCE: Abnormally high hepatic Cu is prevalent in hepatic biopsies from dogs. Machine learning modeling showed that necroinflammation, not cholestasis or cirrhosis, on hepatic histopathology, is predictive of higher hepatic Cu and might be a reliable histologic predictor of CAH.

Oral Vaccination Using a Probiotic Vaccine Platform Combined with Prebiotics Impacts Immune Response and the Microbiome.

Unique to mucosal vaccination is the reciprocal influence of the microbiome and mucosal immune responses, where the immune system is constantly balancing between the clearance of pathogens and the tolerance of self-antigen, food, and the microbiota. Secretory IgA plays a major role in maintaining the homeostasis of a healthy gut microbiome. Natural polyreactive IgA often coats members of the commensal microbiota to aid in their colonization, while high-affinity specific IgA binds to pathogens resulting in their clearance. We developed a probiotic-based mucosal vaccination platform using the bacterium Lactobacillus acidophilus (rLA) with the potential to influence this balance in the IgA coating. In this study, we sought to determine whether repeated administration of rLA alters the host intestinal microbial community due to the immune response against the rLA vaccine. To address this, IgA-seq was employed to characterize shifts in IgA-bound bacterial populations. Additionally, we determined whether using rice bran as a prebiotic would influence the immunogenicity of the vaccine and/or IgA-bound bacterial populations. Our results show that the prebiotic influenced the kinetics of rLA antibody induction and that the rLA platform did not cause lasting disturbances to the microbiome.

Transitions of foliar mycobiota community and transcriptome in response to pathogenic conifer needle interactions.

Profiling the host-mycobiota interactions in healthy vs. diseased forest ecosystems helps understand the dynamics of understudied yet increasingly important threats to forest health that are emerging due to climate change. We analyzed the structural and functional changes of the mycobiota and the responses of Pinus contorta in the Lophodermella needle cast pathosystem through metabarcoding and metatranscriptomics. When needles transitioned from asymptomatic to symptomatic, dysbiosis of the mycobiota occurred, but with an enrichment of Lophodermella pathogens. Many pathogenicity-related genes were highly expressed by the mycobiota at the necrotrophic phase, showing an active pathogen response that are absent in asymptomatic needles. This study also revealed that Lophodermella spp. are members of a healthy needle mycobiota that have latent lifestyles suggesting that other pine needle pathogens may have similar biology. Interestingly, Pinus contorta upregulated defense genes in healthy needles, indicating response to fungal recognition, while a variety of biotic and abiotic stresses genes were activated in diseased needles. Further investigation to elucidate the possible antagonistic interplay of other biotic members leading to disease progression and/or suppression is warranted. This study provides insights into microbial interactions in non-model pathosystems and contributes to the development of new forest management strategies against emerging latent pathogens.

A Randomized Controlled Trial of Dietary Rice Bran Intake on Microbiota Diversity, Enteric Dysfunction, and Fecal Secretory IgA in Malian and Nicaraguan Infants.

BACKGROUND: Malnutrition and diarrhea are leading causes of death in children aged <5 y. Rice bran is a nutrient-dense prebiotic available globally. OBJECTIVES: The objective of this secondary analysis was to evaluate the effects of daily rice bran supplementation on environmental enteric dysfunction (EED) markers, total fecal secretory IgA (sIgA), and microbiota in infants at high risk of malnutrition. METHODS: Six-month-old Malian and Nicaraguan infants were randomly assigned to control or daily rice bran supplementation cohorts (1 to 5 g/d). Feces were collected monthly for 6 mo to evaluate fecal sIgA, markers of EED, and microbiota diversity. Statistical methods included linear mixed models, generalized mixed models, Spearman correlation, and Wilcoxon rank-sum tests. RESULTS: Six-month-old Malian infants had significantly elevated sIgA (4.0× higher, P < 0.001), fecal myeloperoxidase (31.6× higher, P < 0.001), fecal α1-antitrypsin (1.8× higher, P = 0.006), and lower fecal neopterin (0.13× higher, P < 0.001) than the age-matched Nicaraguan infants. In the Nicaraguan rice bran cohort from 6 to 12 mo of age, there was a significant decrease in sIgA concentrations (0.4×, P < 0.05) and a correlation between sIgA and the EED marker α1-antitrypsin (0.523, P < 0.0001) at 12 mo of age. In Malian infants, daily rice bran ingestion resulted in decreased EED scores (0.71×, P = 0.02) and a stable sIgA concentration over time. The rice bran group of Malian infants also had correlation between sIgA and the EED marker neopterin (0.544, P < 0.001) at 12 mo of age and a significant (P < 0.05) increase in microbiota α-diversity at a younger age (9 mo with rice bran compared with 10 mo in control group), which supports earlier microbiota maturation. CONCLUSIONS: These results support rice bran as a functional food ingredient targeting gut mucosa in children at high-risk of malnutrition.

Litter Commensal Bacteria Can Limit the Horizontal Gene Transfer of Antimicrobial Resistance to Salmonella in Chickens.

Fostering a "balanced" gut microbiome through the administration of beneficial microbes that can competitively exclude pathogens has gained a lot of attention and use in human and animal medicine. However, little is known about how microbes affect the horizontal gene transfer of antimicrobial resistance (AMR). To shed more light on this question, we challenged neonatal broiler chicks raised on reused broiler chicken litter-a complex environment made up of decomposing pine shavings, feces, uric acid, feathers, and feed-with Salmonella enterica serovar Heidelberg (S. Heidelberg), a model pathogen. Neonatal chicks challenged with S. Heidelberg and raised on reused litter were more resistant to S. Heidelberg cecal colonization than chicks grown on fresh litter. Furthermore, chicks grown on reused litter were at a lower risk of colonization with S. Heidelberg strains that encoded AMR on IncI1 plasmids. We used 16S rRNA gene sequencing and shotgun metagenomics to show that the major difference between chicks grown on fresh litter and those grown on reused litter was the microbiome harbored in the litter and ceca. The microbiome of reused litter samples was more uniform and enriched in functional pathways related to the biosynthesis of organic and antimicrobial molecules than that in fresh litter samples. We found that Escherichia coli was the main reservoir of plasmids encoding AMR and that the IncI1 plasmid was maintained at a significantly lower copy per cell in reused litter compared to fresh litter. These findings support the notion that commensal bacteria play an integral role in the horizontal transfer of plasmids encoding AMR to pathogens like Salmonella. IMPORTANCE Antimicrobial resistance spread is a worldwide health challenge, stemming in large part from the ability of microorganisms to share their genetic material through horizontal gene transfer. To address this issue, many countries and international organizations have adopted a One Health approach to curtail the proliferation of antimicrobial-resistant bacteria. This includes the removal and reduction of antibiotics used in food animal production and the development of alternatives to antibiotics. However, there is still a significant knowledge gap in our understanding of how resistance spreads in the absence of antibiotic selection and the role commensal bacteria play in reducing antibiotic resistance transfer. In this study, we show that commensal bacteria play a key role in reducing the horizontal gene transfer of antibiotic resistance to Salmonella, provide the identity of the bacterial species that potentially perform this function in broiler chickens, and also postulate the mechanism involved.

Improved non-redundant species screening panels for benchmarking the performance of new investigational antibacterial candidates against Category A and B priority pathogens.

Background: NIAID has a programme for testing drug candidates against biodefense and emerging bacterial pathogens that uses defined strain panels consisting of standard laboratory reference strains and strains of clinical origin. Objectives: The current studies were performed to assess the activity of standard-of-care drugs, determine benchmark criteria for new investigational antibacterial candidate prioritization and identify reduced non-redundant strain panels for candidate performance classification. Methods: The susceptibilities of each strain in the screening panels to 40 standard-of-care drugs and clinical drug combinations were determined by percentage growth inhibition using multiple concentrations, a method commonly used in efficient high-throughput screening efforts. The drug susceptibility of each strain was categorized based on interpretive criteria to benchmark the activity of each standard-of-care drug and drug combination, followed by confirmation of select active drugs. Exact match and clustering analyses defined focused non-redundant species and pan-species screening panels. Results: This process revealed a broad spectrum of susceptibilities among strains in each species, with important differences between the standard laboratory reference strains and strains of clinical origin. Exact match and clustering analyses identified subsets of non-redundant strains that can more efficiently classify drug activity resulting in individual species screening panels, a pan-species screening panel and a pan-species maximum resistance panel. Conclusions: This study resulted in improved non-redundant species screening panels for benchmarking the performance of new investigational antibacterial candidates with the greatest potential for efficacy against clinically relevant Category A and B priority and emerging pathogens.

Accounting for animal movement improves vaccination strategies against wildlife disease in heterogeneous landscapes.

Oral baiting is used to deliver vaccines to wildlife to prevent, control, and eliminate infectious diseases. A central challenge is how to spatially distribute baits to maximize encounters by target animal populations, particularly in urban and suburban areas where wildlife such as raccoons (Procyon lotor) are abundant and baits are delivered along roads. Methods from movement ecology that quantify movement and habitat selection could help to optimize baiting strategies by more effectively targeting wildlife populations across space. We developed a spatially explicit, individual-based model of raccoon movement and oral rabies vaccine seroconversion to examine whether and when baiting strategies that match raccoon movement patterns perform better than currently used baiting strategies in an oral rabies vaccination zone in greater Burlington, Vermont, USA. Habitat selection patterns estimated from locally radio-collared raccoons were used to parameterize movement simulations. We then used our simulations to estimate raccoon population rabies seroprevalence under currently used baiting strategies (actual baiting) relative to habitat selection-based baiting strategies (habitat baiting). We conducted simulations on the Burlington landscape and artificial landscapes that varied in heterogeneity relative to Burlington in the proportion and patch size of preferred habitats. We found that the benefits of habitat baiting strongly depended on the magnitude and variability of raccoon habitat selection and the degree of landscape heterogeneity within the baiting area. Habitat baiting improved seroprevalence over actual baiting for raccoons characterized as habitat specialists but not for raccoons that displayed weak habitat selection similar to radiocollared individuals, except when baits were delivered off roads where preferred habitat coverage and complexity was more pronounced. In contrast, in artificial landscapes with either more strongly juxtaposed favored habitats and/or higher proportions of favored habitats, habitat baiting performed better than actual baiting, even when raccoons displayed weak habitat preferences and where baiting was constrained to roads. Our results suggest that habitat selection-based baiting could increase raccoon population seroprevalence in urban-suburban areas, where practical, given the heterogeneity and availability of preferred habitat types in those areas. Our novel simulation approach provides a flexible framework to test alternative baiting strategies in multiclass landscapes to optimize bait-distribution strategies.