Complete genome sequences of two avian pathogenic Escherichia coli strains isolated from broilers exhibiting colibacillosis in Mississippi.

We report the whole-genome sequences of Escherichia coli strains APEC-O2-MS1266 and APEC-O2-MS1657 isolated from the liver and heart of infected broilers in Mississippi State, US. The genomic information of these two causative strains may provide a valuable reference for comparative studies of avian pathogenic E. coli.

Changes in gene expression in the intestinal mucus of broilers with woody breast myopathy.

Previous work has shown that dietary treatments affect woody breast (WB) incidence differently, which indicates that gut conditions such as gut barrier function, inflammation, and oxidative stress are likely related to WB. In this study, dietary supplementation with antibiotics (bacitracin) or probiotics (Bacillus subtilis) was investigated for their effects on the expression of transcripts related to gut barrier function, inflammation, and oxidative stress in the mucus lining of the jejunum from broilers with or without WB. A split-plot experimental design was used in this study. The dietary treatments served as the main plot factor and the breast muscle condition was the subplot factor. On d 41, jejunum mucus was collected from 1 bird from each of 3 replicate pens in each 3 dietary treatment groups that exhibited WB and an additional bird that contained a normal breast (3 biological replicates/treatment/phenotype; 3 × 3 × 2, total N = 18). Total RNA was extracted using a commercial RNA extraction kit. The expression levels of CLDN1, MUC6, TLR2A, TLR2B, TLR4, IFN-γ, IL-1ß, IL-8L1, IL-10, NOS2, and SOD were determined using 2-step RT-qPCR analysis. The gene expression difference in ΔCt values was determined after normalizing with the chicken 18S rRNA gene. When the significant differences occurred between treatments, the relative fold change was calculated using the ΔΔCt method and the significance level was calculated. The PROC GLM procedure of SAS 9.4 was used, and the level of significance was set at P ≤ 0.05. There were no significant interactive effects between diet and the breast muscle condition on the expression of any of the genes tested. However, birds with WB exhibited higher MUC6 (P < 0.0001) gene expression levels than birds with normal breast muscles. In addition, the expression of SOD decreased in birds that were fed the antibiotic diet when compared to birds that were fed the probiotic diet (P = 0.014). In conclusion, WB identified in broilers tested in the current study is attributed to increased expression of mucin, indicating a correlation between WB incidence and gel-forming mucin secretion and pathogen signaling.

In silico prediction and expression analysis of vaccine candidate genes of Campylobacter jejuni.

Campylobacter jejuni (C. jejuni) is the most common food-borne pathogen that causes human gastroenteritis in the United States. Consumption of contaminated poultry products is considered as the major source of human Campylobacter infection. An effective vaccine would be a promising alternative to antibiotic supplements to curb C. jejuni colonization in poultry gastrointestinal (GI) tract. However, the genetic diversity among the C. jejuni isolates makes vaccine production more challenging. Despite many attempts, an effective Campylobacter vaccine is not yet available. This study aimed to identify suitable candidates to develop a subunit vaccine against C. jejuni, which could reduce colonization in the GI tract of the poultry. In the current study, 4 C. jejuni strains were isolated from retail chicken meat and poultry litter samples and their genomes were sequenced utilizing next-generation sequencing technology. The genomic sequences of C. jejuni strains were screened to identify potential antigens utilizing the reverse vaccinology approach. In silico genome analysis predicted 3 conserved potential vaccine candidates (phospholipase A [PldA], TonB dependent vitamin B12 transporter [BtuB], and cytolethal distending toxin subunit B [CdtB]) suitable for the development of a vaccine. Furthermore, the expression of predicted genes during host-pathogen interaction was analyzed by an infection study using an avian macrophage-like immortalized cell line (HD11). The HD11 was infected with C. jejuni strains, and the RT-qPCR assay was performed to determine the expression of the predicted genes. The expression difference was analyzed using ΔΔCt methods. The results indicate that all 3 predicted genes, PldA, BtuB, and CdtB, were upregulated in 4 tested C. jejuni strains irrespective of their sources of isolation. In conclusion, in silico prediction and gene expression analysis during host-pathogen interactions identified 3 potential vaccine candidates for C. jejuni.

A high-quality chromosome-level genome assembly of rohu carp, Labeo rohita, and its utilization in SNP-based exploration of gene flow and sex determination.

Labeo rohita (rohu) is a carp important to aquaculture in South Asia, with a production volume close to Atlantic salmon. While genetic improvements to rohu are ongoing, the genomic methods commonly used in other aquaculture improvement programs have historically been precluded in rohu, partially due to the lack of a high-quality reference genome. Here we present a high-quality de novo genome produced using a combination of next-generation sequencing technologies, resulting in a 946 Mb genome consisting of 25 chromosomes and 2,844 unplaced scaffolds. Notably, while approximately half the size of the existing genome sequence, our genome represents 97.9% of the genome size newly estimated here using flow cytometry. Sequencing from 120 individuals was used in conjunction with this genome to predict the population structure, diversity, and divergence in three major rivers (Jamuna, Padma, and Halda), in addition to infer a likely sex determination mechism in rohu. These results demonstrate the utility of the new rohu genome in modernizing some aspects of rohu genetic improvement programs.

Innovations in double digest restriction-site associated DNA sequencing (ddRAD-Seq) method for more efficient SNP identification.

We present an improved ddRAD-Seq protocol for identifying single nucleotide polymorphisms (SNPs). It utilizes selected restriction enzyme digestion fragments, quick acting ligases that are neutral with the restriction enzyme buffer eliminating buffer exchange steps, and adapters designed to be compatible with Illumina index primers. Library amplification and barcoding are completed in one PCR step, and magnetic beads are used to purify the genomic fragments from the ligation and library generation steps. Our protocol increases the efficiency and decreases the time to complete a ddRAD-Seq experiment. To demonstrate its utility, we compared SNPs from our protocol with those from whole genome resequencing data from Gossypium herbaceum and Gossypium arboreum. Principal component analysis demonstrated that the variability of the combined data was explained by the genotype (PC1) and methodology applied (PC2). Phylogenetic analysis showed that the SNPs from our method clustered with SNPs from the resequencing data of the corresponding genotype. Sequence alignments illustrated that for homozygous loci, more than 90% of the SNPs from the resequencing data were discovered by our method. Our analyses suggest that our ddRAD-Seq method is reliable in identifying SNPs suitable for phylogenetic and association genetic studies while reducing cost and time over known methods.

Complete Genome Sequences of Four Campylobacter jejuni Strains Isolated from Retail Chicken Meat and Broiler Feces.

Campylobacter jejuni is the leading pathogen that causes foodborne infections. Here, we report the complete genome sequences of four C. jejuni strains isolated from retail chicken meat and broiler feces samples. Genes encoding type VI secretion and antibiotic resistance were detected among these isolates.

Effects of dietary bacitracin or Bacillus subtilis on the woody breast myopathy-associated gut microbiome of Eimeria spp. challenged and unchallenged broilers.

Study suggested that dysbiosis of the gut microbiota may affect the etiology of woody breast (WB). In the current study, the cecal microbiota and WB in chickens fed three different diets were investigated. A total of 504 male chicks were used in a randomized complete block design with a 3 (Diet) × 2 (Challenge) factorial arrangement of treatments with 6 replicates per treatment, 6 treatments per block, and 14 birds per treatment. The experimental diets were a control diet (corn-soybean meal basal diet), an antibiotic diet (basal diet + 6.075 mg bacitracin/kg feed), and a probiotic diet (basal diet + 2.2 × 108 CFU Bacillus subtilis PB6/kg feed). On d 14, birds that were assigned to the challenge treatment received a 20 × live cocci vaccine. On d 41, breast muscle hardness in live birds was palpated and grouped into normal (NB) and WB phenotypes. Cecal contents were collected and their bacterial compositions were analyzed and compared. The genomic DNA of the cecal contents was extracted and the V3 and V4 regions of 16S rRNA gene were amplified and sequenced via an Illumina MiSeq platform. There were no differences (P > 0.05) in Shannon and Chao 1 indexes between the challenges, diets, and phenotypes (NB vs. WB). However, there was a difference (P = 0.001) in the beta diversity of the samples between the challenged and nonchallenged groups. Relative bacterial abundance differed (false discovery rate, FDR < 0.05) between the challenge treatments, but there were no significant differences (FDR > 0.05) among the three diets or two phenotypes. Predicted energy metabolism, nucleotide metabolism, and amino acid and coenzyme biosynthesis activities only differed (q-value < 0.05) between challenged and nonchallenged groups. The cocci challenge altered the gut microbial composition on Butyricicoccus pullicaecorum, Sporobacter termitidis, and Subdoligranulum variabile, but the dietary antibiotic and probiotic treatments did not impact gut microbial composition. No strong association was found between WB myopathy and gut microbial composition in this study.

Functional Diversification of Populus FLOWERING LOCUS D-LIKE3 Transcription Factor and Two Paralogs in Shoot Ontogeny, Flowering, and Vegetative Phenology.

Both the evolution of tree taxa and whole-genome duplication (WGD) have occurred many times during angiosperm evolution. Transcription factors are preferentially retained following WGD suggesting that functional divergence of duplicates could contribute to traits distinctive to the tree growth habit. We used gain- and loss-of-function transgenics, photoperiod treatments, and circannual expression studies in adult trees to study the diversification of three Populus FLOWERING LOCUS D-LIKE (FDL) genes encoding bZIP transcription factors. Expression patterns and transgenic studies indicate that FDL2.2 promotes flowering and that FDL1 and FDL3 function in different vegetative phenophases. Study of dominant repressor FDL versions indicates that the FDL proteins are partially equivalent in their ability to alter shoot growth. Like its paralogs, FDL3 overexpression delays short day-induced growth cessation, but also induces distinct heterochronic shifts in shoot development-more rapid phytomer initiation and coordinated delay in both leaf expansion and the transition to secondary growth in long days, but not in short days. Our results indicate that both regulatory and protein coding sequence variation contributed to diversification of FDL paralogs that has led to a degree of specialization in multiple developmental processes important for trees and their local adaptation.

The identification of type I MADS box genes as the upstream activators of an endosperm-specific invertase inhibitor in Arabidopsis.

BACKGROUND: Nuclear endosperm development is a common mechanism among Angiosperms, including Arabidopsis. During nuclear development, the endosperm nuclei divide rapidly after fertilization without cytokinesis to enter the syncytial phase, which is then followed by the cellularized phase. The endosperm can be divided into three spatial domains with distinct functions: the micropylar, peripheral, and chalazal domains. Previously, we identified two putative small invertase inhibitors, InvINH1 and InvINH2, that are specifically expressed in the micropylar region of the syncytial endosperm. In addition, ectopically expressing InvINH1 in the cellularized endosperm led to a reduction in embryo growth rate. However, it is not clear what are the upstream regulators responsible for the specific expression of InvINHs in the syncytial endosperm. RESULTS: Using protoplast transient expression system, we discovered that a group of type I MADS box transcription factors can form dimers to activate InvINH1 promoter. Promoter deletion assays carried out in the protoplast system revealed the presence of an enhancer region in InvINH1 promoter, which contains several consensus cis-elements for the MADS box proteins. Using promoter deletion assay in planta, we further demonstrated that this enhancer region is required for InvINH1 expression in the syncytial endosperm. One of the MADS box genes, AGL62, is a key transcription factor required for syncytial endosperm development. Using promoter-GFP reporter assay, we demonstrated that InvINH1 and InvINH2 are not expressed in agl62 mutant seeds. Collectively, our data supports the role of AGL62 and other type I MADS box genes as the upstream activators of InvINHs expression in the syncytial endosperm. CONCLUSIONS: Our findings revealed several type I MADS box genes that are responsible for activating InvINH1 in the syncytial endosperm, which in turn regulates embryo growth rate during early stage of seed development.

Complete genome sequence of multidrug-resistant avian pathogenic Escherichia coli strain APEC-O2-MS1170.

OBJECTIVES: Avian pathogenic Escherichia coli (APEC) causes colibacillosis, one of the leading causes of mortality and morbidity associated with significant economic losses in the poultry industry. This study aimed to determine antimicrobial resistance and to characterise the genome sequence of a multidrug-resistant (MDR) APEC strain isolated from a broiler chicken. METHODS: Strain APEC-O2-MS1170 was isolated from the broiler yolk sac of a 14-day-old broiler. Antimicrobial susceptibility testing was performed using a Sensititre National Antimicrobial Resistance Monitoring System (NARMS) Gram-negative panel. Whole-genome sequencing was performed using both the long-read sequencing approach with a Nanopore GridION sequencer and short-read sequencing with an Illumina HiSeq X-Ten sequencer to obtain a complete scaffold of the genome and an accurate sequence. RESULTS: The genome size of strain APEC-O2-MS1170 is 4,993,909 bp with a GC content of 50.7% and 4,651 protein-coding sequences. Public databases were used to identify the virulence-associated gene and antimicrobial resistance gene cargo. Plasmid comparison showed that pAPEC-O2-MS1170-R is a large multidrug resistance IncB/O/K/Z plasmid, while pAPEC-O2-MS1170-ColV shares homology with the APEC ColV virulence plasmid. CONCLUSION: The genome sequence of APEC-O2-MS1170 provides valuable information on resistance mechanisms and virulence characteristics of pathogenic E. coli as well as information for tracing the potential spread of this MDR strain.

Identification of Antimicrobial Resistance Determinants in Aeromonas veronii Strain MS-17-88 Recovered From Channel Catfish (Ictalurus punctatus).

Aeromonas veronii is a Gram-negative species ubiquitous in different aquatic environments and capable of causing a variety of diseases to a broad host range. Aeromonas species have the capability to carry and acquire antimicrobial resistance (AMR) elements, and currently multi-drug resistant (MDR) Aeromonas isolates are commonly found across the world. A. veronii strain MS-17-88 is a MDR strain isolated from catfish in the southeastern United States. The present study was undertaken to uncover the mechanism of resistance in MDR A. veronii strain MS-17-88 through the detection of genomic features. To achieve this, genomic DNA was extracted, sequenced, and assembled. The A. veronii strain MS-17-88 genome comprised 5,178,226-bp with 58.6% G+C, and it encoded several AMR elements, including imiS, ampS, mcr-7.1, mcr-3, catB2, catB7, catB1, floR, vat(F), tet(34), tet(35), tet(E), dfrA3, and tetR. The phylogeny and resistance profile of a large collection of A. veronii strains, including MS-17-88, were evaluated. Phylogenetic analysis showed a close relationship between MS-17-88 and strain Ae5 isolated from fish in China and ARB3 strain isolated from pond water in Japan, indicating a common ancestor of these strains. Analysis of phage elements revealed 58 intact, 63 incomplete, and 15 questionable phage elements among the 53 A. veronii genomes. The average phage element number is 2.56 per genome, and strain MS-17-88 is one of two strains having the maximum number of identified prophage elements (6 elements each). The profile of resistance against various antibiotics across the 53 A. veronii genomes revealed the presence of tet(34), mcr-7.1, mcr-3, and dfrA3 in all genomes (100%). By comparison, sul1 and sul2 were detected in 7.5% and 1.8% of A. veronii genomes. Nearly 77% of strains carried tet(E), and 7.5% of strains carried floR. This result suggested a low abundance and prevalence of sulfonamide and florfenicol resistance genes compared with tetracycline resistance among A. veronii strains. Overall, the present study provides insights into the resistance patterns among 53 A. veronii genomes, which can inform therapeutic options for fish affected by A. veronii.

Identification and characterization of microRNAs (miRNAs) and their transposable element origins in the saltwater crocodile, Crocodylus porosus.

MicroRNAs (miRNAs) are 18-24 nucleotide regulatory RNAs. They are involved in the regulation of genetic and biological pathways through post transcriptional gene silencing and/or translational repression. Data suggests a slow evolutionary rate for the saltwater crocodile (Crocodylus porosus) over the past several million years when compared to birds, the closest extant relatives of crocodilians. Understanding gene regulation in the saltwater crocodile in the context of relatively slow genomic change thus holds potential for the investigation of genomics, evolution, and adaptation. Utilizing eleven tissue types and sixteen small RNA libraries, we report 644 miRNAs in the saltwater crocodile with >78% of miRNAs being novel to crocodilians. We also identified potential targets for the miRNAs and analyzed the relationship of the miRNA repertoire to transposable elements (TEs). Results suggest an increased association of DNA transposons with miRNAs when compared to retrotransposons. This work reports the first comprehensive analysis of miRNAs in Crocodylus porosus and addresses the potential impacts of miRNAs in regulating the genome in the saltwater crocodile. In addition, the data suggests a supporting role of TEs as a source for miRNAs, adding to the increasing evidence that TEs play a significant role in the evolution of gene regulation.

Altered expression of lactate dehydrogenase and monocarboxylate transporter involved in lactate metabolism in broiler wooden breast.

Wooden breast (WB) results in significant losses to the broiler industry due to reductions in meat quality. While the etiology of WB is unknown, it is believed to be associated with localized hypoxia and decreased lactate levels in skeletal muscles, indicating the presence of altered lactate metabolism in WB. We hypothesized that the expression levels of the major signaling molecules that control lactate metabolism, including lactate dehydrogenases (LDHA and LDHB) and monocarboxylate transporters (MCT1 and MCT4), were altered in WB. Therefore, the objectives of this study were to evaluate whether there were changes in mRNA and protein levels of LDHA, LDHB, MCT1, and MCT4 in WB compared to normal breast (NB) muscles. Biochemical analysis for LDH enzyme activity in NB and WB muscles was studied. MicroRNA375 (miR-375) expression, known to be inversely associated with LDHB protein expression in human cells, was also investigated. The level of LDHA mRNA was 1.7-fold lower in WB tissues than in NB tissues (P < 0.0001). However, the LDHA protein levels were similar in WB and NB tissues. In contrast, the levels of LDHB mRNA and protein were 8.4-fold higher (P < 0.002) and 13.6-fold higher (P < 0.02) in WB than in NB tissues, respectively. The level of miR-375 was not different between WB and NB muscles. The specific LDH isoenzyme activity that converted lactate to pyruvate was 1.8-fold lower in WB compared to NB tissues (P < 0.01). The level of MCT1 mRNA was 2.3-fold higher in WB than those in NB muscles (P < 0.02). However, this upregulation was not observed with MCT1 protein expression levels. The expression levels of MCT4 mRNA and protein were elevated 2.8-fold (P < 0.02) and 3.5-fold (P < 0.004) in WB compared to NB tissues, respectively. Our current findings suggest the potential roles of LDHB and MCT4 on lactate metabolism and provide a unique molecular elucidation for altered lactate homeostasis in WB muscles of broilers.

Publisher Correction: Therapeutic potential of N-acetylcysteine in acrylamide acute neurotoxicity in adult zebrafish.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Targeting redox metabolism: the perfect storm induced by acrylamide poisoning in the brain.

Exposure to acrylamide may lead to different neurotoxic effects in humans and in experimental animals. To gain insights into this poorly understood type of neurotoxicological damage, we used a multi-omic approach to characterize the molecular changes occurring in the zebrafish brain exposed to acrylamide at metabolite, transcript and protein levels. We detected the formation of acrylamide adducts with thiol groups from both metabolites and protein residues, leading to a quasi-complete depletion of glutathione and to the inactivation of different components of the thioredoxin system. We propose that the combined loss-of-function of both redox metabolism-related systems configure a perfect storm that explains many acrylamide neurotoxic effects, like the dysregulation of genes related to microtubules, presynaptic vesicle alteration, and behavioral alterations. We consider that our mechanistical approach may help developing new treatments against the neurotoxic effects of acrylamide and of other neurotoxicants that may share its toxic mode of action.

Gut microbiota and short chain fatty acid composition as affected by legume type and processing methods as assessed by simulated in vitro digestion assays.

This study's objective was to investigate how legume type and processing method affected digestibility, and subsequent gut microbiota and short chain fatty acid (SCFA) formation. After autoclaving and germinating-cooking, pinto bean and soybean were subjected to in vitro digestion. The digestion residues were fractionated into soluble and insoluble fiber, and fermented by microbiota from pig feces. Results showed the in vitro digestibility was affected significantly by processing method and legume type. Autoclaving resulted in higher digestibility. The in-vitro digested bean residues caused a rapid pH decrease in the first 12 h during the fermentation with pig feces, and a significant increse in the formation of SCFAs. A positive modulation of the gut microbiota by the in-vitro digested bean residues was observed. Prevotella copri and Bacteroides vulgatus exhibited the highest relative abundance in the treatments with germinated bean's soluble residues. Phascolarctobacterium succinatutens was increased by the insoluble residues.

Molecular evolution in immune genes across the avian tree of life.

All organisms encounter pathogens, and birds are especially susceptible to infection by malaria parasites and other haemosporidians. It is important to understand how immune genes, primarily innate immune genes which are the first line of host defense, have evolved across birds, a highly diverse group of tetrapods. Here, we find that innate immune genes are highly conserved across the avian tree of life and that although most show evidence of positive or diversifying selection within specific lineages or clades, the number of sites is often proportionally low in this broader context of putative constraint. Rather, the evidence shows a much higher level of negative or purifying selection in these innate immune genes - rather than adaptive immune genes - which is consistent with birds' long coevolutionary history with pathogens and the need to maintain a rapid response to infection. We further explored avian responses to haemosporidians by comparing differential gene expression in wild birds (1) uninfected with haemosporidians, (2) infected with Plasmodium, and (3) infected with Haemoproteus (Parahaemoproteus). We found patterns of significant differential expression with some genes unique to infection with each genus and a few shared between "treatment" groups, but none that overlapped with the genes included in the phylogenetic study.

Therapeutic potential of N-acetylcysteine in acrylamide acute neurotoxicity in adult zebrafish.

Two essential key events in acrylamide (ACR) acute neurotoxicity are the formation of adducts with nucleophilic sulfhydryl groups on cysteine residues of selected proteins in the synaptic terminals and the depletion of the glutathione (GSx) stores in neural tissue. The use of N-acetylcysteine (NAC) has been recently proposed as a potential antidote against ACR neurotoxicity, as this chemical is not only a well-known precursor of the reduced form of glutathione (GSH), but also is an scavenger of soft electrophiles such as ACR. In this study, the suitability of 0.3 and 0.75 mM NAC to protect against the neurotoxic effect of 0.75 mM ACR has been tested in vivo in adult zebrafish. NAC provided only a mild to negligible protection against the changes induced by ACR in the motor function, behavior, transcriptome and proteome. The permeability of NAC to cross blood-brain barrier (BBB) was assessed, as well as the ACR-scavenging activity and the gamma-glutamyl-cysteine ligase (γ-GCL) and acylase I activities. The results show that ACR not only depletes GSx levels but also inhibits it synthesis from NAC/cysteine, having a dramatic effect over the glutathione system. Moreover, results indicate a very low NAC uptake to the brain, probably by a combination of low BBB permeability and high deacylation of NAC during the intestinal absorption. These results strongly suggest that the use of NAC is not indicated in ACR acute neurotoxicity treatment.

A genomic resource for the sedentary semi-endoparasitic reniform nematode, Rotylenchulus reniformis Linford & Oliveira.

The reniform nematode (Rotylenchulus reniformis) is a sedentary semi-endoparasitic species that is pathogenic on many row crops, fruits, and vegetables. Here, the authors present a draft genome assembly of R. reniformis using small- and large-insert libraries sequenced on the Illumina GAIIx and MiSeq platforms.The reniform nematode (Rotylenchulus reniformis) is a sedentary semi-endoparasitic species that is pathogenic on many row crops, fruits, and vegetables. Here, the authors present a draft genome assembly of R. reniformis using small- and large-insert libraries sequenced on the Illumina GAIIx and MiSeq platforms.

Complete Genome Sequence of Multidrug-Resistant Edwardsiella ictaluri Strain MS-17-156.

Edwardsiella ictaluri is a significant pathogen of cultured fish, particularly channel catfish. Here, we present the complete genome sequence of a multidrug-resistant E. ictaluri strain, MS-17-156, isolated from diseased channel catfish. The genome sequence of this multidrug-resistant strain is expected to help us understand the molecular mechanism of antibiotic resistance in this important pathogen.