Prevalence and molecular characterization of Salmonella isolated from wild birds in fresh produce environments.

Wild birds pose a difficult food safety risk to manage because they can avoid traditional wildlife mitigation strategies, such as fences. Birds often use agricultural fields and structures as foraging and nesting areas, which can lead to defecation on crops and subsequent transfer of foodborne pathogens. To assess the food safety risk associated with these events, wild bird feces were collected from produce fields across the southeastern United States during the 2021 and 2022 growing seasons. In total 773 fecal samples were collected from 45 farms across Florida, Georgia, South Carolina, and Tennessee, and 2.1% (n = 16) of samples were Salmonella-positive. Importantly, 75% of Salmonella were isolated from moist feces, showing reduced Salmonella viability when feces dry out. 16S microbiome analysis showed that presence of culturable Salmonella in moist feces correlated to a higher proportion of the Enterobacteriaceae family. From the Salmonella-positive samples, 62.5% (10/16) contained multi-serovar Salmonella populations. Overall, 13 serovars were detected, including six most commonly attributed to human illness (Enteriditis, Newport, Typhimurium, Infantis, Saintpaul, and Muenchen). PCR screening identified an additional 59 Salmonella-positive fecal samples, which were distributed across moist (n = 44) and dried feces (n = 15). On-farm point counts and molecular identification from fecal samples identified 57 bird species, including for 10 Salmonella-positive fecal samples. Overall, there was a low prevalence of Salmonella in fecal samples, especially in dried feces, and we found no evidence of Salmonella transmission to proximal foliage or produce. Fecal samples collected in farms close together shared highly related isolates by whole genome sequencing and also had highly similar Salmonella populations with comparable relative frequencies of the same serovars, suggesting the birds acquired Salmonella from a common source.

Evidence for common ancestry and microevolution of passerine-adapted Salmonella enterica serovar Typhimurium in the UK and USA.

The evolution of Salmonella enterica serovar Typhimurium (S. Typhimurium) within passerines has resulted in pathoadaptation of this serovar to the avian host in Europe. Recently, we identified an S. Typhimurium lineage from passerines in North America. The emergence of passerine-adapted S. Typhimurium in Europe and North America raises questions regarding its evolutionary origin. Here, we demonstrated that the UK and US passerine-adapted S. Typhimurium shared a common ancestor from ca. 1838, and larids played a key role in the clonal expansion by disseminating the common ancestor between North America and Europe. Further, we identified virulence gene signatures common in the passerine- and larid-adapted S. Typhimurium, including conserved pseudogenes in fimbrial gene lpfD and Type 3 Secretion System (T3SS) effector gene steC. However, the UK and US passerine-adapted S. Typhimurium also possessed unique virulence gene signatures (i.e. pseudogenes in fimbrial gene fimC and T3SS effector genes sspH2, gogB, sseJ and sseK2), and the majority of them (38/47) lost a virulence plasmid pSLT that was present in the larid-adapted S. Typhimurium. These results provide evidence that passerine-adapted S. Typhimurium share a common ancestor with those from larids, and the divergence of passerine- and larid-adapted S. Typhimurium might be due to pseudogenization or loss of specific virulence genes.

Salmonella enterica Serovar Typhimurium Isolates from Wild Birds in the United States Represent Distinct Lineages Defined by Bird Type.

Salmonella enterica serovar Typhimurium is typically considered a host generalist; however, certain isolates are associated with specific hosts and show genetic features of host adaptation. Here, we sequenced 131 S. Typhimurium isolates from wild birds collected in 30 U.S. states during 1978-2019. We found that isolates from broad taxonomic host groups including passerine birds, water birds (Aequornithes), and larids (gulls and terns) represented three distinct lineages and certain S. Typhimurium CRISPR types presented in individual lineages. We also showed that lineages formed by wild bird isolates differed from most isolates originating from domestic animal sources, and that genomes from these lineages substantially improved source attribution of Typhimurium genomes to wild birds by a machine learning classifier. Furthermore, virulence gene signatures that differentiated S. Typhimurium from passerines, water birds, and larids were detected. Passerine isolates tended to lack S. Typhimurium-specific virulence plasmids. Isolates from the passerine, water bird, and larid lineages had close genetic relatedness with human clinical isolates, including those from a 2021 U.S. outbreak linked to passerine birds. These observations indicate that S. Typhimurium from wild birds in the United States are likely host-adapted, and the representative genomic data set examined in this study can improve source prediction and facilitate outbreak investigation. IMPORTANCE Within-host evolution of S. Typhimurium may lead to pathovars adapted to specific hosts. Here, we report the emergence of disparate avian S. Typhimurium lineages with distinct virulence gene signatures. The findings highlight the importance of wild birds as a reservoir for S. Typhimurium and contribute to our understanding of the genetic diversity of S. Typhimurium from wild birds. Our study indicates that S. Typhimurium may have undergone adaptive evolution within wild birds in the United States. The representative S. Typhimurium genomes from wild birds, together with the virulence gene signatures identified in these bird isolates, are valuable for S. Typhimurium source attribution and epidemiological surveillance.

Methods for simultaneous determination of legacy and insensitive munition (IM) constituents in aqueous, soil/sediment, and tissue matrices.

Currently, no standard method exists for analyzing insensitive munition (IM) compounds in environmental matrices, with or without concurrent legacy munition compounds, resulting in potentially inaccurate determinations. The primary objective of this work was to develop new methods of extraction, pre-concentration, and analytical separation/quantitation of 17 legacy munition compounds along with several additional IM compounds, IM breakdown products, and other munition compounds that are not currently included in U. S. Environmental Protection Agency (EPA) Method 8330B. The eight additional compounds included were nitroguanidine, 3-nitro-1,2,4-triazol-5-one, picric acid, 2,4-dinitroanisole, 2,4-dinitrophenol, 2-nitrophenol, 4-nitrophenol, and new surrogate ortho-nitrobenzoic acid (o-NBA). Analytical methods were developed to enable sensitive, simultaneous detection and quantitation of the 24 IM and legacy compounds, including two orthogonal high-performance liquid chromatography (HPLC) column separations with either ultraviolet (UV) or mass spectrometric (MS) detection. Procedures were developed for simultaneous extraction of all 24 analytes and two surrogates (1,2-dinitrobenzene, 1,2-DNB; o-NBA) from high- and low-level aqueous matrices and solid matrices, using acidification, solid phase extraction (SPE), or solvent extraction (SE), respectively. For low-level aqueous samples extracted by SPE, all compounds were recovered within current Department of Defense Quality Systems Manual (DoD QSM) Ver5.3 accepted limits for aqueous samples analyzed by EPA Method 8330B (57-135%), except NQ, which was consistently recovered at approximately 50%. Likewise, all compounds were recovered from six geographically/geochemically unique soil types within current QSM accepted limits for solid samples analyzed by EPA Method 8330B (64-135%). Further, the majority of compounds were recovered from four tissue types within current limits for solids, with generally low recovery only for Tetryl (from 4 to 62%). A preparatory chromatographic interference removal procedure was adapted for tissue extracts, as various analytical interferences were observed for all studied tissue types.

Effects of acute exposures of 2,4,6-trinitrotoluene and inorganic lead on the fecal microbiome of the green anole (Anolis carolinensis).

Microbiome studies focused on ecologically relevant vertebrate models like reptiles have been limited. Because of their relatively small home range, fast maturation, and high fecundity, lizards are an excellent reptilian terrestrial indicator species. For this study we used the green anole, Anolis carolinensis, to assess the impact of military relevant contaminants on fecal microbiome composition. Fourteen day sub-acute exposures were conducted via oral gavage with 2,4,6-Trinitrotoluene (TNT) and inorganic lead at doses of 60 mg/kg and 20 mg/kg of body weight, respectively. Body weights and food consumption were monitored and fecal samples were collected for high-throughput 16S rRNA gene amplicon sequencing and analytical chemistry at days 0 and 15. At the end of the study, liver and gut were harvested for body burden data. Chemical analysis confirmed accumulation of TNT, TNT transformation products, and lead in liver tissue and fecal samples. Bacterial community analysis of fecal material revealed significant differences between day 0 and day 15 of TNT exposed anoles with an operational taxonomic unit (OTU) within the genus Erwinia representing 32% of the microbial community in TNT exposed anoles. Predictable changes in gut microbiome composition could offer an easily assayed, noninvasive biomarker for specific chemical exposure providing enhanced scientific support to risk assessments on military installations.

Accumulation of 2,4-dinitroanisole in the earthworm Eisenia fetida from chemically spiked and aged natural soils.

An initiative within the US military is targeting the replacement of traditional munitions constituents with insensitive munitions to reduce the risk of accidental detonation. The bioavailability and bioaccumulative potential of the insensitive munitions compound 2,4-dinitroanisole (DNAN) to Eisenia fetida was assessed in soils with different geochemical characteristics. Prior to exposure, soils were chemically spiked with DNAN and aged for 1 wk or 29 wk. Transformation products 2- and 4-amino-nitroanisole (2A-4NAN and 4A-2NAN) occurred in aged soils and their porewater but never at concentrations higher than the residual DNAN. The sum of DNAN, 2A-4NAN, and 4A-2NAN (sumDNAN) in soil decreased with aging, likely by irreversible binding. Both clay and organic matter contents of the soil appeared to affect the bioavailability of DNAN. The sumDNAN body residues of earthworms approached apparent steady state after 1 d and remained relatively constant through to day 7. Higher concentrations of 2A-4NAN and 4A-2NAN measured in worm tissues relative to those in soil suggest reductive transformation of DNAN in the tissues. Mean bioaccumulation factors (ratio of tissue to soil concentrations) varied from 1.2 to 4.3, whereas mean bioconcentration factors (ratio of tissue to porewater concentrations) ranged from 1.4 to 3.2. Porewater seems to play a significant role in the accumulation of DNAN in earthworms, consistent with equilibrium partitioning theory. The concentration of DNAN in soil porewater could serve as an indicator of bioavailability as well as a predictor of the concentration of that compound in earthworms. Environ Toxicol Chem 2016;35:1835-1842. Publlished 2015 SETAC. This article is a US Government work, and as such, is in the public domain in the United States of America.