Survival of Antimicrobial Resistant Salmonella Heidelberg Inoculated into Microcosms of Fresh Pine Wood Shavings for Broiler Litter.

This study applied genomic characterizations to a cocktail of three Salmonella enterica serovar Heidelberg (S. Heidelberg) strains different antimicrobial resistance (AMR) profile which were inoculated into fresh pine wood shavings (PWS) microcosms. The strains were isolated from feces (SH-AAFC), carcass (SH-ARS) and thigh (SH-FSIS) of broiler chicken. SH-AAFC harbored an antimicrobial resistant gene (ARG) blaCMY-2 on an IncI1 plasmid while SH-FSIS harbored multiple ARGs (floR, cmlA1, tet(A), blaTEM-1B, ant(2'')-Ia, aph(6)-Id, aph(3'')-Ib and sul2) on an IncC plasmid. SH-ARS was pan-susceptible. The die-off of Salmonella was determined at days 0, 1, 7, 14 and 21. Antibiotic susceptibility tests and whole genome sequencing were performed on 77 isolates. At 21 days post-inoculation, Salmonella abundance decreased by 4.4 Log10 CFU/g with the water activity of PWS being correlated with Salmonella survival. SH-AAFC clonal populations survived longer in PWS than SH-FSIS and SH-ARS populations. SH-AAFC clones persisting in litter carried higher copy number of Col plasmids than their ancestors, while some SH-ARS clones acquired a lysogenic bacteriophage from SH-FSIS populations. These results suggests that mobile genetic determinants such as plasmids (which could carry ARGs) and bacteriophage plays roles in the persistence of S. Heidelberg in the PWS used as broiler litter.

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.

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.

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.

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.

An Open-Source Program (Haplo-ST) for Whole-Genome Sequence Typing Shows Extensive Diversity among Listeria monocytogenes Isolates in Outdoor Environments and Poultry Processing Plants.

A reliable and standardized classification of Listeria monocytogenes is important for accurate strain identification during outbreak investigations. Current whole-genome sequencing (WGS)-based approaches for strain characterization are either difficult to standardize, rendering them less suitable for data exchange, or are not freely available. Thus, we developed a portable and open-source tool, Haplo-ST, to improve standardization and provide maximum discriminatory potential to WGS data tied to a multilocus sequence typing (MLST) framework. Haplo-ST performs whole-genome MLST (wgMLST) for L. monocytogenes while allowing for data exchangeability worldwide. This tool takes in (i) raw WGS reads as input, (ii) cleans the raw data according to user-specified parameters, (iii) assembles genes across loci by mapping to genes from reference strains, and (iv) assigns allelic profiles to assembled genes and provides a wgMLST subtyping for each isolate. Data exchangeability relies on the tool assigning allelic profiles based on a centralized nomenclature defined by the widely used BIGSdb-Lm database. Tests of Haplo-ST's performance with simulated reads from L. monocytogenes reference strains demonstrated high sensitivity (97.5%), and coverage depths of ≥20× were found to be sufficient for wgMLST profiling. We then used Haplo-ST to characterize and differentiate between two groups of L. monocytogenes isolates derived from the natural environment and poultry processing plants. Phylogenetic reconstruction identified lineages within each group, and no lineage specificity was observed with isolate phenotypes (transient versus persistent) or origins. Genetic differentiation analyses between isolate groups identified 21 significantly differentiated loci, potentially enriched for adaptation and persistence of L. monocytogenes within poultry processing plants.IMPORTANCE We have developed an open-source tool (https://github.com/swarnalilouha/Haplo-ST) that provides allele-based subtyping of L. monocytogenes isolates at the whole-genome level. Along with allelic profiles, this tool also generates allele sequences and identifies paralogs, which is useful for phylogenetic tree reconstruction and deciphering relationships between closely related isolates. More broadly, Haplo-ST is flexible and can be adapted to characterize the genome of any haploid organism simply by installing an organism-specific gene database. Haplo-ST also allows for scalable subtyping of isolates; fewer reference genes can be used for low-resolution typing, whereas higher resolution can be achieved by increasing the number of genes used in the analysis. Our tool enabled clustering of L. monocytogenes isolates into lineages and detection of potential loci for adaptation and persistence in food processing environments. Findings from these analyses highlight the effectiveness of Haplo-ST in subtyping and evaluating relationships among isolates in studies of bacterial population genetics.

Horizontal Gene Transfer and Acquired Antibiotic Resistance in Salmonella enterica Serovar Heidelberg following In Vitro Incubation in Broiler Ceca.

The chicken gastrointestinal tract harbors microorganisms that play a role in the health and disease status of the host. The cecum is the part of the gut that carries the highest microbial densities, has the longest residence time of digesta, and is a vital site for urea recycling and water regulation. Therefore, the cecum provides a rich environment for bacteria to horizontally transfer genes between one another via mobile genetic elements such as plasmids and bacteriophages. In this study, we used broiler chicken cecum as a model to investigate antibiotic resistance genes that can be transferred in vitro from cecal flora to Salmonella enterica serovar Heidelberg. We used whole-genome sequencing and resistome enrichment to decipher the interactions between S Heidelberg, the gut microbiome, and acquired antibiotic resistance. After 48 h of incubation of ceca under microaerophilic conditions, we recovered one S Heidelberg isolate with an acquired IncK2 plasmid (88 kb) carrying an extended-spectrum-ß-lactamase gene (blaCMY-2). In vitro, this plasmid was transferable between Escherichia coli and S Heidelberg strains but transfer was unsuccessful between S Heidelberg strains. An in-depth genetic characterization of transferred plasmids suggests that they share significant homology with P1-like phages. This study contributes to our understanding of horizontal gene transfer between an important foodborne pathogen and the chicken gut microbiome.IMPORTANCES. Heidelberg is a clinically important serovar, linked to foodborne illness and among the top 5 serovars isolated from poultry in the United States and Canada. Acquisition of new genetic material from the microbial flora in the gastrointestinal tract of food animals, including broilers, may contribute to increased fitness of pathogens like S. Heidelberg and may increase their level of antibiotic tolerance. Therefore, it is critical to gain a better understanding of the interactions that occur between important pathogens and the commensals present in the animal gut and other agroecosystems. In this report, we show that the native flora in broiler ceca were capable of transferring mobile genetic elements carrying the AmpC ß-lactamase (blaCMY-2) gene to an important foodborne pathogen, S Heidelberg. The potential role for bacteriophage transduction is also discussed.

MEGARes: an antimicrobial resistance database for high throughput sequencing.

Antimicrobial resistance has become an imminent concern for public health. As methods for detection and characterization of antimicrobial resistance move from targeted culture and polymerase chain reaction to high throughput metagenomics, appropriate resources for the analysis of large-scale data are required. Currently, antimicrobial resistance databases are tailored to smaller-scale, functional profiling of genes using highly descriptive annotations. Such characteristics do not facilitate the analysis of large-scale, ecological sequence datasets such as those produced with the use of metagenomics for surveillance. In order to overcome these limitations, we present MEGARes (https://megares.meglab.org), a hand-curated antimicrobial resistance database and annotation structure that provides a foundation for the development of high throughput acyclical classifiers and hierarchical statistical analysis of big data. MEGARes can be browsed as a stand-alone resource through the website or can be easily integrated into sequence analysis pipelines through download. Also via the website, we provide documentation for AmrPlusPlus, a user-friendly Galaxy pipeline for the analysis of high throughput sequencing data that is pre-packaged for use with the MEGARes database.

Determining the distribution of fitness effects using a generalized Beta-Burr distribution.

In Beisel et al. (2007), a likelihood framework, based on extreme value theory (EVT), was developed for determining the distribution of fitness effects for adaptive mutations. In this paper we extend this framework beyond the extreme distributions and develop a likelihood framework for testing whether or not extreme value theory applies. By making two simple adjustments to the Generalized Pareto Distribution (GPD) we introduce a new simple five parameter probability density function that incorporates nearly every common (continuous) probability model ever used. This means that all of the common models are nested. This has important implications in model selection beyond determining the distribution of fitness effects. However, we demonstrate the use of this distribution utilizing likelihood ratio testing to evaluate alternative distributions to the Gumbel and Weibull domains of attraction of fitness effects. We use a bootstrap strategy, utilizing importance sampling, to determine where in the parameter space will the test be most powerful in detecting deviations from these domains and at what sample size, with focus on small sample sizes (n<20). Our results indicate that the likelihood ratio test is most powerful in detecting deviation from the Gumbel domain when the shape parameters of the model are small while the test is more powerful in detecting deviations from the Weibull domain when these parameters are large. As expected, an increase in sample size improves the power of the test. This improvement is observed to occur quickly with sample size n≥10 in tests related to the Gumbel domain and n≥15 in the case of the Weibull domain. This manuscript is in tribute to the contributions of Dr. Paul Joyce to the areas of Population Genetics, Probability Theory and Mathematical Statistics. A Tribute section is provided at the end that includes Paul's original writing in the first iterations of this manuscript. The Introduction and Alternatives to the GPD sections were obtained from the last iteration that Paul and I have worked on, with some adjustments. I hope that the rest gives justice to his thoughts and possible conclusions that he might have wanted to portray.

Maize death acids, 9-lipoxygenase-derived cyclopente(a)nones, display activity as cytotoxic phytoalexins and transcriptional mediators.

Plant damage promotes the interaction of lipoxygenases (LOXs) with fatty acids yielding 9-hydroperoxides, 13-hydroperoxides, and complex arrays of oxylipins. The action of 13-LOX on linolenic acid enables production of 12-oxo-phytodienoic acid (12-OPDA) and its downstream products, termed "jasmonates." As signals, jasmonates have related yet distinct roles in the regulation of plant resistance against insect and pathogen attack. A similar pathway involving 9-LOX activity on linolenic and linoleic acid leads to the 12-OPDA positional isomer, 10-oxo-11-phytodienoic acid (10-OPDA) and 10-oxo-11-phytoenoic acid (10-OPEA), respectively; however, physiological roles for 9-LOX cyclopentenones have remained unclear. In developing maize (Zea mays) leaves, southern leaf blight (Cochliobolus heterostrophus) infection results in dying necrotic tissue and the localized accumulation of 10-OPEA, 10-OPDA, and a series of related 14- and 12-carbon metabolites, collectively termed "death acids." 10-OPEA accumulation becomes wound inducible within fungal-infected tissues and at physiologically relevant concentrations acts as a phytoalexin by suppressing the growth of fungi and herbivores including Aspergillus flavus, Fusarium verticillioides, and Helicoverpa zea. Unlike previously established maize phytoalexins, 10-OPEA and 10-OPDA display significant phytotoxicity. Both 12-OPDA and 10-OPEA promote the transcription of defense genes encoding glutathione S transferases, cytochrome P450s, and pathogenesis-related proteins. In contrast, 10-OPEA only weakly promotes the accumulation of multiple protease inhibitor transcripts. Consistent with a role in dying tissue, 10-OPEA application promotes cysteine protease activation and cell death, which is inhibited by overexpression of the cysteine protease inhibitor maize cystatin-9. Unlike jasmonates, functions for 10-OPEA and associated death acids are consistent with specialized roles in local defense reactions.

Derivation of chicken induced pluripotent stem cells tolerant to Newcastle disease virus-induced lysis through multiple rounds of infection.

BACKGROUND: Newcastle disease (ND), caused by Newcastle disease virus (NDV), is a devastating disease of poultry and wild birds. ND is prevented by rigorous biocontainment and vaccination. One potential approach to prevent spread of the virus is production of birds that show innate resistance to NDV-caused disease. Induced pluripotent stem cell (iPSC) technology allows adult cells to be reprogrammed into an embryonic stem cell-like state capable of contributing to live offspring and passing on unique traits in a number of species. Recently, iPSC approaches have been successfully applied to avian cells. If chicken induced pluripotent stem cells (ciPSCs) are genetically or epigenetically modified to resist NDV infection, it may be possible to generate ND resistant poultry. There is limited information on the potential of ciPSCs to be infected by NDV, or the capacity of these cells to become resistant to infection. The aim of the present work was to assess the characteristics of the interaction between NDV and ciPSCs, and to develop a selection method that would increase tolerance of these cells to NDV-induced cellular damage. RESULTS: Results showed that ciPSCs were permissive to infection with NDV, and susceptible to virus-mediated cell death. Since ciPSCs that survived infection demonstrated the ability to recover quickly, we devised a system to select surviving cells through multiple infection rounds with NDV. ciPSCs that sustained 9 consecutive infections had a statistically significant increase in survival (up to 36 times) compared to never-infected ciPSCs upon NDV infection (tolerant cells). Increased survival was not caused by a loss of permissiveness to NDV replication. RNA sequencing followed by enrichment pathway analysis showed that numerous metabolic pathways where differentially regulated between tolerant and never-infected ciPSCs. CONCLUSIONS: Results demonstrate that ciPSCs are permissive to NDV infection and become increasingly tolerant to NDV under selective pressure, indicating that this system could be applied to study mechanisms of cellular tolerance to NDV.

Subtyping of Salmonella enterica Subspecies I Using Single-Nucleotide Polymorphisms in Adenylate Cyclase.

Methods to rapidly identify serotypes of Salmonella enterica subspecies I are of vital importance for protecting the safety of food. To supplement the serotyping method dkgB-linked intergenic sequence ribotyping (ISR), single-nucleotide polymorphisms were characterized within adenylate cyclase (cyaA). The National Center for Biotechnology Information (NCBI) database had 378 cyaA sequences from S. enterica subspecies I, which included 42 unique DNA sequences and 19 different amino acid sequences. Five representative isolates, namely serotypes Typhimurium, Kentucky, Enteritidis phage type PT4, and two variants of Enteritidis phage type PT13a, were differentiated within a microsphere-based fluidics system in cyaA by allele-specific primer extension. Validation against 25 poultry-related environmental Salmonella isolates representing 11 serotypes yielded a ∼89% success rate at identifying the serotype of the isolate, and a different region could be targeted to achieve 100%. When coupled with ISR, all serotypes were differentiated. Phage lineages of serotype Enteritidis 13a and 4 were identified, and a biofilm-forming strain of PT13a was differentiated from a smooth phenotype within phage type. Comparative ranking of mutation indices to genes such as the tRNA transferases, the diguanylate cyclases, and genes used for multilocus sequence typing indicated that cyaA is an appropriate gene for assessing epidemiological trends of Salmonella because of its relative stability in nucleotide composition.

Evaluation of disinfectants to prevent mechanical transmission of viruses and a viroid in greenhouse tomato production.

BACKGROUND: In recent years, a number of serious disease outbreaks caused by viruses and viroids on greenhouse tomatoes in North America have resulted in significant economic losses to growers. The objectives of this study were to evaluate the effectiveness of commercial disinfectants against mechanical transmission of these pathogens, and to select disinfectants with broad spectrum reactivity to control general virus and viroid diseases in greenhouse tomato production. METHODS: A total of 16 disinfectants were evaluated against Pepino mosaic virus (PepMV), Potato spindle tuber viroid (PSTVd), Tomato mosaic virus (ToMV), and Tobacco mosaic virus (TMV). The efficacy of each disinfectant to deactivate the pathogen's infectivity was evaluated in replicate experiments from at least three independent experiments. Any infectivity that remained in the treated solutions was assessed through bioassays on susceptible tomato plants through mechanical inoculation using inocula that had been exposed with the individual disinfectant for three short time periods (0-10 sec, 30 sec and 60 sec). A positive infection on the inoculated plant was determined through symptom observation and confirmed with enzyme-linked immunosorbent assay (PepMV, ToMV, and TMV) and real-time reverse transcription-PCR (PSTVd). Experimental data were analyzed using Logistic regression and the Bayesian methodology. RESULTS: Statistical analyses using logistic regression and the Bayesian methodology indicated that two disinfectants (2% Virkon S and 10% Clorox regular bleach) were the most effective to prevent transmission of PepMV, PSTVd, ToMV, and TMV from mechanical inoculation. Lysol all-purpose cleaner (50%) and nonfat dry milk (20%) were also effective against ToMV and TMV, but with only partial effects for PepMV and PSTVd. CONCLUSION: With the broad spectrum efficacy against three common viruses and a viroid, several disinfectants, including 2% Virkon S, 10% Clorox regular bleach and 20% nonfat dry milk, are recommend to greenhouse facilities for consideration to prevent general virus and viroid infection on tomato plants.

Comparison of Resistance to Asiatic Citrus Canker Among Different Genotypes of Citrus in a Long-Term Canker-Resistance Field Screening Experiment in Brazil.

Asiatic citrus canker (ACC) is caused by Xanthomonas citri subsp. citri. The disease results in yield loss and renders fruit unfit for the fresh market. A 6-year study in Paraná State, Brazil, was conducted to compare the susceptibility of 186 genotypes of citrus representing sweet orange (Citrus sinensis), mandarin (C. reticulata), Mediterranean mandarin (C. deliciosa), Clementine mandarin (C. clementina), Satsuma mandarin (C. unshiu), sour orange (C. aurantium), lemon (C. limon), sweet lime (C. aurantifolia), grapefruit (C. paradisi), and four hybrids (C. reticulata × Citrus sp., C. reticulata × C. paradisi, C. reticulata × C. sinensis, and C. unshiu × C. sinensis). Sweet orange (C. sinensis) was represented by the most genotypes (n = 141). The number of lesions per leaf was assessed 18 times from 2005 to 2010 (up to 4 times per year). The data were analyzed using mixed-model analysis of fixed and random effects, which showed a total of six resistance-susceptibility groupings of species and hybrids. Based on species, the most resistant genotypes, on average, included Satsuma and lemon (mean lesions per leaf = 4.32 and 4.26, respectively), and the most susceptible genotypes were grapefruit and sweet lime, with 14.84 and 10.96 lesions per leaf, respectively. Genotypes of mandarin, sour orange, Mediterranean mandarin, and sweet orange had intermediate severity (5.48 to 9.56 lesions per leaf). The hybrids also showed a range of ACC severity but all were in the more resistant groupings (5.26 to 7.35 lesions per leaf). No genotype was immune to ACC. The most resistant genotype was 'Muscia' (C. reticulata) and the most susceptible was 'Valencia Frost' (C. sinensis) (1.86 and 14.78 lesions per leaf, respectively). Approximately one-sixth of the genotypes showed a negative relationship of mean lesions per leaf with time, suggesting increasing resistance as they aged, due to a reduction in either new flush or plant size and structure. These results of the relative susceptibility of different citrus genotypes can be used in future research and to assist in varietal selection or for breeding purposes both within Brazil and other regions where ACC is an issue.

Mi-1-Mediated Nematode Resistance in Tomatoes is Broken by Short-Term Heat Stress but Recovers Over Time.

Tomato (Solanum lycopersicum L.) is among the most valuable agricultural products, but Meloidogyne spp. (root-knot nematode) infestations result in serious crop losses. In tomato, resistance to root-knot nematodes is controlled by the gene Mi-1, but heat stress interferes with Mi-1-associated resistance. Inconsistent results in published field and greenhouse experiments led us to test the effect of short-term midday heat stress on tomato susceptibility to Meloidogyne incognita race 1. Under controlled day/night temperatures of 25°C/21°C, 'Amelia', which was verified as possessing the Mi-1 gene, was deemed resistant (4.1 ± 0.4 galls/plant) and Rutgers, which does not possess the Mi-1 gene, was susceptible (132 ± 9.9 galls/plant) to M. incognita infection. Exposure to a single 3 hr heat spike of 35°C was sufficient to increase the susceptibility of 'Amelia' but did not affect Rutgers. Despite this change in resistance, Mi-1 gene expression was not affected by heat treatment, or nematode infection. The heat-induced breakdown of Mi-1 resistance in 'Amelia' did recover with time regardless of additional heat exposures and M. incognita infection. These findings would aid in the development of management strategies to protect the tomato crop at times of heightened M. incognita susceptibility.

Vaginal microbiome of reproductive-age women.

The means by which vaginal microbiomes help prevent urogenital diseases in women and maintain health are poorly understood. To gain insight into this, the vaginal bacterial communities of 396 asymptomatic North American women who represented four ethnic groups (white, black, Hispanic, and Asian) were sampled and the species composition characterized by pyrosequencing of barcoded 16S rRNA genes. The communities clustered into five groups: four were dominated by Lactobacillus iners, L. crispatus, L. gasseri, or L. jensenii, whereas the fifth had lower proportions of lactic acid bacteria and higher proportions of strictly anaerobic organisms, indicating that a potential key ecological function, the production of lactic acid, seems to be conserved in all communities. The proportions of each community group varied among the four ethnic groups, and these differences were statistically significant [χ(2)(10) = 36.8, P < 0.0001]. Moreover, the vaginal pH of women in different ethnic groups also differed and was higher in Hispanic (pH 5.0 ± 0.59) and black (pH 4.7 ± 1.04) women as compared with Asian (pH 4.4 ± 0.59) and white (pH 4.2 ± 0.3) women. Phylotypes with correlated relative abundances were found in all communities, and these patterns were associated with either high or low Nugent scores, which are used as a factor for the diagnosis of bacterial vaginosis. The inherent differences within and between women in different ethnic groups strongly argues for a more refined definition of the kinds of bacterial communities normally found in healthy women and the need to appreciate differences between individuals so they can be taken into account in risk assessment and disease diagnosis.

Host Functional Response to a Prototypic Orally Delivered Self-Replicating Vaccine Platform.

The development of mucosal vaccines has been limited and could be aided by a systems vaccinology approach to identify platforms and adjuvant strategies that induce protective immune responses. The induction of local immune responses by mucosal-delivered vaccines has been difficult to evaluate from peripheral samples, as systemic responses often do not correlate with the mucosal response. Here, we utilized transcriptomics in combination with Gene Set Enrichment Analysis (GSEA) to assess innate immune activation by an oral probiotic Lactobacillus acidophilus-based vaccine platform in mice. The goal was to explore the earliest immune responses elicited after oral immunization at the Peyer's patch. Twenty-four hours after oral delivery of the L. acidophilus vaccine platform, we found an abundance of L. acidophilus at Peyer's patches and detected expression of the vaccine viral proteins and adjuvants, confirming in vivo vaccine delivery. Compared to mice orally dosed with buffer or wild-type L. acidophilus, we identified enhanced responses in immune pathways related to cytokine and gene signaling, T and B cell activation, phagocytosis, and humoral responses. While more work is needed to correlate these pathways with protection from infection and/or disease, they indicate this method's potential to evaluate and aid in the iterative development of next-generation mucosal vaccines.

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.

Impacts of Antiretroviral Therapy on the Oral Microbiome and Periodontal Health of Feline Immunodeficiency Virus Positive Cats.

Feline immunodeficiency virus (FIV) is the domestic cat analogue of HIV infection in humans. Both viruses induce oral disease in untreated individuals, with clinical signs that include gingivitis and periodontal lesions. Oral disease manifestations in HIV patients are abated by highly effective combination antiretroviral therapy (cART), though certain oral manifestations persist despite therapy. Microorganisms associated with oral cavity opportunistic infections in patients with HIV cause similar pathologies in cats. To further develop this model, we evaluated characteristics of feline oral health and oral microbiome during experimental FIV infection over an 8-month period following cART. Using 16S metagenomics sequencing, we evaluated gingival bacterial communities at four timepoints in uninfected and FIV-infected cats treated with cART or placebo. Comprehensive oral examinations were also conducted by a veterinary dental specialist over the experimental period. Gingival inflammation was higher in FIV-infected cats treated with placebo compared to cART-treated cats and controls at study endpoint. Oral microbiome alpha diversity increased in all groups, while beta diversity differed among treatment groups, documenting a significant effect of cART therapy on microbiome community composition. This finding has not previously been reported and indicates cART ameliorates immunodeficiency virus-associated oral disease via preservation of oral mucosal microbiota. Further, this study illustrates the value of the FIV animal model for investigations of mechanistic associations and therapeutic interventions for HIV oral manifestations.

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.