Staphylococcus agnetis is an emerging pathogen in chickens but has been most commonly isolated from sub-clinical mastitis in bovines. Previous whole-genome analyses for known virulence genes failed ...to identify determinants for the switch from mild ductal infections in cattle to severe infections in poultry. We now report identification of a family of 15 kbp, 17-19 gene mobile genetic elements (MGEs) specific to chicken osteomyelitis and dermatitis isolates of S. agnetis. These MGEs can be present in multiple copies per genome. The MGE has been vectored on a Staphylococcus phage that separately lysogenized two S. agnetis osteomyelitis strains. The S. agnetis genome from a broiler breeder case of ulcerative dermatitis contains 2 orthologs of this MGE, not associated with a prophage. BLASTn and phylogenetic analyses show that there are closely related intact MGEs found in genomes of S. aureus. The genome from a 1980s isolate from chickens in Ireland contains 3 copies of this MGE. More recent chicken isolates descended from that genome (Poland 2009, Oklahoma 2010, and Arkansas 2018) contain 2 to 4 related copies. Many of the genes of this MGE can be identified in disparate regions of the genomes of other chicken isolates of S. aureus. BLAST searches of the NCBI databases detect no similar MGEs outside of S. aureus and S. agnetis. These MGEs encode no proteins related to those produced by Staphylococcus aureus Pathogenicity Islands, which have been associated with the transition of S. aureus from human to chicken hosts. Other than mobilization functions, most of the genes in these new MGEs annotate as hypothetical proteins. The MGEs we describe appear to represent a new family of Chromosomal Islands (CIs) shared amongst S. agnetis and S. aureus. Further work is needed to understand the role of these CIs/MGEs in pathogenesis. Analysis of horizontal transfer of genetic elements between isolates and species of Staphylococci provides clues to evolution of host-pathogen interactions as well as revealing critical determinants for animal welfare and human diseases.
Abstract Scorpions, a seemingly primitive, stinging arthropod taxa, are known to exhibit marked diversity in their venom components. These venoms are known for their human pathology, but they are ...also important as models for therapeutic and drug development applications. In this study, we report a high-quality genome assembly and annotation of the striped bark scorpion, Centruroides vittatus, created with several shotgun libraries. The final assembly is 760 Mb in size, with a BUSCO score of 97.8%, a 30.85% GC, and an N50 of 2.35 Mb. We estimated 36,189 proteins with 37.32% assigned to Gene Ontology (GO) terms in our GO annotation analysis. We mapped venom toxin genes to 18 contigs and 2 scaffolds. We were also able to identify expression differences between venom gland (telson) and body tissue (carapace) with 19 sodium toxin and 14 potassium toxin genes to 18 contigs and 2 scaffolds. This assembly, along with our transcriptomic data, provides further data to investigate scorpion venom genomics.
We investigated lameness outbreaks at 3 commercial broiler farms in Arkansas. We isolated several distinct bacterial species from Bacterial Chondronecrosis with Osteomyelitis (BCO) lesions from these ...3 farms. The results show that BCO-lameness pathogens on particular farms can differ significantly. We characterized genomes for isolates of the 2 most prevalent species, Escherichia coli and Staphylococcus aureus. Genomes assembled for E. coli isolates from all 3 farms were quite different between farms, and most similar to genomes from different geographical locations and hosts. The E. coli phylogenomics suggests frequent host shifts for this species. Genomes for S. aureus isolates from one farm were highly related to those from chicken isolates from Europe. Highly related isolates have also been characterized from chickens in the Arkansas area for at least a decade. Phylogenomics suggest that this S. aureus has been restricted to poultry for more than 40 y. Detailed analysis of genomes from 2 neighboring clades of S. aureus human and chicken isolates, identifies the acquisition of a specific pathogenicity island in the transition from human to chicken pathogen and that pathogenesis for this clade in chickens may depend on this mobile element. Investigation of the evolution of this chicken-restricted clade from 1980 in Ireland, Poland in 2008, Oklahoma in 2010 and Arkansas in 2019, reveals the acquisition of additional virulence determinants including pathogenicity islands. Isolate-specific genome characterizations will help further our understanding of the disease mechanisms of BCO-lameness, a significant animal welfare issue.
We used an embryo lethality assay (ELA) to assess virulence for different isolates from cases of bacterial chondronecrosis with osteomyelitis (BCO) in broilers. Lameness is among the most significant ...animal welfare issues in the poultry industry. Bacterial infections are a major cause of lameness and different bacterial species have been obtained from lame broilers. Reliable lab-based assays are required to assess relative virulence of bacteria obtained from lame broilers. ELA has been used to assess lethal dosage of Enterococcus faecalis and Enterococcus cecorum. We hypothesized that ELA could substitute for more laborious and costly assessments of BCO isolate pathogenicity using live birds. We evaluated 2 different levels of bacteria injected into eggs from layer and commercial broiler embryos. Significant findings include 1) Escherichia coli from neighboring farms operated by the same integrator had very different embryo lethality, 2) isolate Staphylococcus agnetis 908 had low virulence in ELA, even though this isolate can induce more than 50% BCO lameness, 3) Enterococcus cecorum 1415 also had low pathogenicity; even though it was recovered from severe bilateral tibial dyschondroplasia, 4) human and chicken BCO isolates of S. aureus had significant pathogenicity, 5) virulence for some isolates was highly variable possibly corresponding with quality of the embryos/fertile eggs used, and 6) ELA pathogenicity was much lower for our BCO isolates than previous reports which may reflect maternal environment. Overall, ELA virulence and BCO virulence are not always concordant indicating that ELA may not be an effective measure for assessing virulence with respect to BCO.
Scorpion systematics and taxonomy have recently shown a need for revision, partially due to insights from molecular techniques. Scorpion taxonomy has been difficult with morphological characters as ...disagreement exists among researchers with character choice for adequate species delimitation in taxonomic studies. Within the family Buthidae, species identification and delimitation is particularly difficult due to the morphological similarity among species and extensive intraspecific morphological diversity. The genus Centruroides in the western hemisphere is a prime example of the difficulty in untangling the taxonomic complexity within buthid scorpions. In this paper, we present phylogeographic, Ecological Niche Modeling, and morphometric analyses to further understand how population diversification may have produced morphological diversity in Centruroides vittatus (Say, 1821). We show that C. vittatus populations in the Big Bend and Trans-Pecos region of Texas, USA are phylogeographically distinct and may predate the Last Glacial Maximum (LGM). In addition, we suggest the extended isolation of Big Bend region populations may have created the C. vittatus variant once known as C. pantheriensis.
This report demonstrates that high levels of lameness can be induced by a limited bacterial challenge in drinking water for birds raised on litter flooring, comparable with lameness induced by the ...gold standard for inducing lameness, growth on suspended wire flooring. The bacterium used in the challenge was cultured from lesions in birds induced for bacterial chondronecrosis with osteomyelitis (BCO) in the wire-flooring model so the epidemiology appears similar. The litter-flooring model could better approximate broiler operations. Furthermore, the work demonstrates that 2 commercial probiotics (GalliProTect and GalliProMax) can reduce lameness in the bacterial challenge litter-flooring model. Lameness attributable to BCO is one of the most significant animal welfare issues for broiler production. The wire-flooring and litter-flooring models afford alternatives for understanding the etiology, and epidemiology of BCO, and development of management strategies to reduce lameness. Probiotics afford a promising management strategy. The results suggest that the probiotic protection may extend beyond just intestinal health and intestinal barrier function.
DNA extraction for downstream molecular diagnostic applications can be an expensive, time-consuming process. We devised a method to quickly extract total bacterial DNA from environmental samples ...based on the sodium hydroxide lysis of cells with or without capture by magnetic beads for subsequent PCR or quantitative PCR. The final DNA extraction method using NaOH is extremely low-cost and can be completed in as little as 10 min at room temperature with dilution, or the DNA can be further purified using silica-coated paramagnetic beads. NaOH extraction was effective for Gram-negative and Gram-positive bacteria in samples from air, soil, sewage, food, laboratory surfaces, and chicken cloacal swabs. The NaOH extraction method was comparable to commercial kits for extraction of DNA from pig fecal samples for 16S amplicon sequencing analyses. We demonstrated that an impinger and portable pump can efficiently capture bacteria from the air in poultry facilities for rapid NaOH extraction to quantify total bacteria and for detection of specific species using qPCR. The air sampling and NaOH extraction procedures are well suited for routine, high-throughput screening and for metagenomic analyses for specific pathogens, even in resource-limited situations.
Ascites syndrome is the most severe manifestation of pulmonary hypertension in fast-growing broilers. The disease can be attributed to increased body weights of birds, where the higher metabolic load ...is not matched by sufficient oxygen supply to the cells and tissues. Although there are environmental components, the disease exhibits moderate to high heritability. The current study uses high throughput whole genome resequencing (WGR) to identify genes and chromosomal regions associated with ascites.
The WGR data identified the CPQ gene on chromosome 2. The association was confirmed by genotyping a large collection of DNAs from phenotyped birds from three distinct broiler lines using SNPs in intron 6 and exon 8 of the CPQ gene. By combining the genotype data for these two SNP loci, we identified three different alleles segregating in the three broiler lines. Particular genotypes could be associated with resistance to ascites. We further determined that particular genotypes most associated with resistance overexpress CPQ mRNA in three tissues which might explain the role of these alleles in contributing to resistance.
Our findings indicate CPQ is an important determinant of pulmonary hypertension syndrome leading to ascites in broilers. We identified particular SNPs that can be used for marker-assisted selection of broilers for resistance to the disease. Our findings validate WGR as a highly efficient approach to map determinants contributing to complex phenotypic or disease-related traits. The CPQ gene has been associated with pulmonary hypertension in genome-wide association studies in humans. Therefore, ascites investigations in broilers are likely to provide insights into some forms of hypertension in humans.
Lameness in broiler chickens is a significant animal welfare and financial issue. Lameness can be enhanced by rearing young broilers on wire flooring. We have identified Staphylococcus agnetis as ...significantly involved in bacterial chondronecrosis with osteomyelitis (BCO) in proximal tibia and femorae, leading to lameness in broiler chickens in the wire floor system. Administration of S. agnetis in water induces lameness. Previously reported in some cases of cattle mastitis, this is the first report of this poorly described pathogen in chickens. We used long and short read next generation sequencing to assemble single finished contigs for the genome and a large plasmid from the chicken pathogen. Comparison of the S. agnetis genome to those of other pathogenic Staphylococci shows that S.agnetis contains a distinct repertoire of virulence determinants. Additionally, the S. agnetis genome has several regions that differ substantially from the genomes of other pathogenic Staphylococci. Comparison of our finished genome to a recent draft genome for a cattle mastitis isolate suggests that future investigations focus on the evolutionary epidemiology of this emerging pathogen of domestic animals.
The feed additive Availa-ZMC was investigated for the ability to reduce lameness in broilers using 2 alternative models for inducing lameness. The mixture of organic trace minerals was effective in ...reducing lameness by 20% in the wire flooring model and 25% in the litter flooring model with the bacterial challenge. Lameness in both models is overwhelmingly attributable to bacterial chondronecrosis with osteomyelitis. The reduction in lameness was associated, at least in part, with enhanced intestinal barrier integrity mediated by elevated expression of tight junction proteins and stimulation of bactericidal killing of adherent peripheral blood monocytes obtained from the birds treated with Availa-ZMC. Lameness is a major animal welfare concern in broiler production. The wire flooring model and litter flooring model with the bacterial challenge are effective models for evaluation of management strategies for mitigating infectious causes of lameness.
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