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Influenza viruses are contagious zoonotic pathogens that belong to the Orthomyxoviridae family, which consists of 4 genera: Alphainfluenzavirus (influenza A virus), Betainfluenzavirus (influenza B virus), Gammainfluenzavirus (influenza C virus [ICV]), [...]

Co-products from the ethanol industry, such as distillers dried grains with solubles (DDGS), can provide alternative protein sources for pet food. Corn fermented protein (CFP) is produced using postfermentation technology to split the protein and yeast from fiber prior to drying. This results in a higher protein ingredient compared to DDGS, increasing its appeal for pet food. In addition, the substantial yeast component, at approximately 20% to 25%, may promote gut health through modulation of the microbiome and the production of short-chain fatty acids. Therefore, the objective of this study was to determine the effect of CFP on the fecal microbiome of cats. The 4 experimental diets included a control with no yeast (T1) and diets containing either 3.5% brewer's dried yeast (T2), 2.5% brewer's dried yeast plus 17.5% DDGS (T3), or 17.5% CFP (T4). All diets except T1 were formulated to contain 3.5% yeast. Diets were fed to adult cats (n  = 11) in an incomplete 4 × 4 replicated Latin square design. Cats were adapted to diet for 9 d followed by a 5-d total fecal collection. During each collection period, fresh fecal samples from each cat were collected and stored at −80 °C until analysis. Fresh fecal samples (n  = 44) were analyzed by 16S rRNA gene sequencing. Raw sequences were processed through Mothur (v.1.44.1). Community diversity was evaluated in R (v4.0.3). Relative abundance was analyzed within the 50 most abundant operational taxonomic unitsusing a mixed model of SAS (v9.4, SAS Institute, Inc. Cary, NC). Diet was the fixed effect and cat and period were random effects. Results were considered significant at P  < 0.05. Alpha-diversity indices (Observed, Chao1, Shannon, Simpson) and beta-diversity metric (principal coordinate analysis) were similar for all treatments. Predominant phyla were Firmicutes (66%), Bacteroidetes (25%), Actinobacteria (8%), Proteobacteria (0.64%), and Desulfobacteria (0.54%). The relative abundance of Firmicutes and Actinobacteria was lower (P  < 0.05) for T3 compared to T4 and T2, respectively. On a more specific phylogenic level, 17 genera resulted in differences (P  < 0.05) among dietary treatments. Overall, this data indicates that compared to traditional yeast and distillers dried grains, CFP did not alter the overall diversity of the fecal microbiome of healthy adult cats over a 14-d period. [ABSTRACT FROM AUTHOR]

Different food processing parameters may alter starch granule structure and its cooking degree. With lower thermomechanical energy, more resistant starch (RS) is retained in the food, which may benefit gastrointestinal (GI) health. The objective of this study was to determine the effect of food processing on dietary utilization and dog gut health. Experimental diets containing 56% corn as the sole starch source were produced through pelleting, baking, and extrusion and compared to a baked control diet in which the corn was replaced with dextrose. The extruded diet resulted in the highest level (P  < 0.05) of in vitro starch cook and lowest RS, while baked was intermediate and pelleted had the lowest starch cook and highest RS. To evaluate the in vivo effects of these treatments, 12 dogs were adapted to foods for 9 d, and feces were collected for 5 d in a replicated 4 × 4 Latin square design. Feces were scored for consistency using an ordinal scale, and parametric data included apparent digestibility (ATTD), parameters indicative of gut health, and the microbial composition, which was centered log-ratio transformed before operational taxonomic unit (OTU) analyses. Fecal scores were analyzed by ordinal logistic regression, and parametric data were analyzed as mixed models. Overall ATTD was greater (P  < 0.05) in extruded, followed by baked and pelleted. Dogs fed the control had osmotic diarrhea, whereas dogs fed the other treatments had mostly acceptable fecal scores, with extrusion leading to the best fecal quality. The control also led to high fecal pH and low SCFAs, indicating dysbiosis. All corn foods had similar (P  > 0.05) fecal SCFAs and extruded tended (P  = 0.055) to promote higher fecal butyrate than baked and pelleted. The microbiome of dogs fed the corn foods had similar α diversity indices, and OTUs at the species and phyla levels were mostly alike and different from the control. In conclusion, the higher levels of in vitro RS did not translate into a better in vivo fermentation profile, and extruded kibble performed best regarding fecal quality, ATTD, and fecal SCFAs. [ABSTRACT FROM AUTHOR]

Simple Summary: Corn-fermented protein, a co-product of ethanol production, can be utilized as a protein source for pet food. Currently, there are no studies that have evaluated the impact of this ingredient on the fecal microbiota of dogs, an indicator of animal health. The overall richness and diversity of the fecal microbiota were maintained when dogs were fed corn-fermented protein compared to traditional ingredients such as brewer's dried yeast and distiller's dried grains with solubles. Corn-fermented protein (CFP), a co-product from the ethanol industry, is produced using post-fermentation technology to split the protein and yeast from fiber prior to drying. The objective of this study was to determine the effect of CFP compared to traditional ingredients on the fecal microbiota of dogs. The four experimental diets included a control with no yeast and diets containing either 3.5% brewer's dried yeast, 2.5% brewer's dried yeast plus 17.5% distiller's dried grains with solubles, or 17.5% CFP. The experimental diets were fed to adult dogs (n = 12) in a 4 × 4 replicated Latin square design. Fresh fecal samples (n = 48) were analyzed by 16S metagenomic sequencing. Raw sequences were processed through mothur. Community diversity was evaluated in R. Relative abundance data were analyzed within the 50 most abundant operational taxonomic units using a mixed model of SAS. Alpha and beta diversity were similar for all treatments. Predominant phyla among all samples were Firmicutes (73%), Bacteroidetes (15%), Fusobacteria (8%), and Actinobacteria (4%). There were no quantifiable (p > 0.05) shifts in the predominant phyla among the treatments. However, nine genera resulted in differences in relative abundance among the treatments. These data indicate that compared to traditional ingredients, CFP did not alter the overall diversity of the fecal microbiota of healthy adult dogs over 14 days. [ABSTRACT FROM AUTHOR]

The SARS‐CoV‐2 virus is the causative agent of COVID‐19 and has undergone continuous mutations throughout the pandemic. The more transmissible Omicron variant has quickly spread and is replacing the Delta variant as the most prevalent strain globally, including in the United States. A new molecular assay that can detect and differentiate both the Delta and Omicron variants was developed. A collection of 660,035 SARS‐CoV‐2 full‐ or near‐full genomes, including 169,454 Delta variant and 24,202 Omicron variant strains, were used for primer and probe designs. In silico data analysis predicted an assay coverage of >99% of all strains, including >99% of the Delta and >99% of Omicron strains. The Omicron variant differential test was designed based on the Δ31‐33 aa deletion in the N‐gene, which is present in the original B.1.1.529 main genotype, BA.1, as well as in BA.2 and BA.3 subtypes. Therefore, the assay should detect the majority of all Omicron variant strains. Standard curves generated with human clinical samples indicated that the PCR amplification efficiencies were 104%, 90.7% and 90.4% for the Omicron, Delta, and non‐Delta/non‐Omicron wild‐type genotypes, respectively. Correlation coefficients of the standard curves were all >0.99. The detection limit of the assay was 14.3, 32.0, and 21.5 copies per PCR reaction for Omicron, Delta, and wild‐type genotypes, respectively. The assay was designed to specifically detect SAR‐CoV‐2 strains. Selected samples with Omicron, Delta and wild‐type genotypes identified by the RT‐qPCR assay were also confirmed by sequencing. The assay did not detect any animal coronavirus‐positive samples that were tested. Human nasal swab samples that previously tested positive (n = 182) or negative (n = 42) for SARS‐CoV‐2 by the ThermoFisher TaqPath COVID‐19 Combo Kit, produced the same result with the new assay. Among positive samples, 55.5% (101/182), 23.1% (42/182), and 21.4% (39/182) were identified as Omicron, Delta, and non‐Omicron/non‐Delta wild‐type genotypes, respectively. [ABSTRACT FROM AUTHOR]

A novel respiratory‐associated Mycoplasma species (M. sp. nov.) of unknown clinical significance was recently identified that causes false positive results with multiple published PCR methods reported to specifically detect Mycoplasma ovipneumonaie, a well‐known respiratory pathogen in small ruminants. This necessitates our objective to develop a real‐time PCR (qPCR) assay for improved specificity and sensitivity, and more rapid detection and differentiation of M. ovipneumoniae and the M. sp. nov. in domestic sheep (DS) and domestic goat (DG) samples, as compared to a conventional PCR and sequencing (cPCR‐seq) assay. Primers and probes were designed based on available M. ovipneumoniae 16S rRNA gene sequences in the GenBank database, and partial 16S rRNA gene sequences provided by the United States Department of Agriculture, Agricultural Research Service (USDA‐ARS) for M. ovipneumoniae and M. sp. nov. USDA‐ARS provided DS (n = 153) and DG (n = 194) nasal swab nucleic acid that previously tested positive for either M. ovipneumoniae (n = 117) or M. sp. nov. (n = 138), or negative for both targets (n = 92) by cPCR‐seq. A host 18S rRNA gene was included as an internal control to monitor for the failure of nucleic acid extraction and possible PCR inhibition. For samples positive by cPCR‐seq, qPCR agreement was 88.0% (103/117; κ = 0.81) and 89.9% (124/138; κ = 0.84) for M. ovipneumoniae and M. sp. nov., respectively; 12 of 255 (4.7%) cPCR‐seq positive samples were qPCR positive for both targets. Of samples negative by cPCR for both mycoplasmas, qPCR detected M. ovipneumoniae and M. sp. nov. in 6.5% (6/92) and 4.3% (4/92), respectively. Samples with discordant results between the cPCR and sequencing assay and the new qPCR were analyzed by target sequencing; successfully sequenced samples had identity matches that confirmed the qPCR result. The increased target specificity of this qPCR is predicted to increase testing accuracy as compared to other published assays. [ABSTRACT FROM AUTHOR]

The Delta variant of SARS‐CoV‐2 has now become the predominant strain in the global COVID‐19 pandemic. Strain coverage of some detection assays developed during the early pandemic stages has declined due to periodic mutations in the viral genome. We have developed a real‐time RT‐PCR (RT‐qPCR) for SARS‐CoV‐2 detection that provides nearly 100% strain coverage, and differentiation of highly transmissible Delta variant strains. All full or nearly full (≥28 kb) SARS‐CoV‐2 genomes (n = 403,812), including 6422 Delta and 280 Omicron variant strains, were collected from public databases at the time of analysis and used for assay design. The two amino acid deletions in the spike gene (S‐gene, Δ156‐157) that is characteristic of the Delta variant were targeted during the assay design. Although strain coverage for the Delta variant was very high (99.7%), detection coverage for non‐Delta wild‐type strains was 93.9%, mainly due to the confined region of design. To increase strain coverage of the assay, the design for CDC N1 target was added to the assay. In silico analysis of 403,812 genomes indicated a 95.4% strain coverage for the CDC N1 target, however, in combination with our new non‐Delta S‐gene target, total coverage for non‐Delta wild‐type strains increased to 99.8%. A human 18S rRNA gene was also analyzed and used as an internal control. The final four‐plex RT‐qPCR assay generated PCR amplification efficiencies between 95.4% and 102.0% with correlation coefficients (R2) of >0.99 for cloned positive controls; Delta and non‐Delta human clinical samples generated PCR efficiencies of 93.4%–97.0% and R2 > 0.99. The assay also detects 98.6% of 280 Omicron sequences. Assay primers and probes have no match to other closely related human coronaviruses, and did not produce a signal from samples positive to selected animal coronaviruses. Genotypes of selected clinical samples identified by the RT‐qPCR were confirmed by Sanger sequencing. [ABSTRACT FROM AUTHOR]

Porcine reproductive and respiratory syndrome (PRRS) remains one of the most economically devastating diseases in swine population in the United States of America. Due to high mutation rate of the PRRS virus (PRRSV) genome, it is difficult to develop an accurate diagnostic assay with high strain coverage. Differentiation of field strains from the four vaccines that have been used in the USA, namely Ingelvac PRRS MLV, Ingelvac ATP, Fostera PRRS and Prime Pac PRRS, adds an additional challenge. It is difficult to use current real‐time PCR systems to detect and differentiate the field strains from the vaccine strains. Luminex xTAG technology allows us to detect more molecular targets in a single reaction with a cost similar to a single real‐time PCR reaction. By analysing all available 678 type 2 PRRSV (PRRSV‐2) complete genome sequences, including the 4 vaccine strains, two pairs of detection primers were designed targeting the conserved regions of ORF4‐ORF7, with strain coverage of 98.8% (670/678) based on in silico analysis. The virus strains sharing ≥98% identity of the complete genomes with the vaccine strains were considered vaccine or vaccine‐like strains. One pair of primers for each vaccine strain were designed targeting the nsp2 region. In silico analysis showed the assay matched 94.7% (54/57) of Ingelvac PRRS® MLV (MLV) strain and the MLV‐like strains, and 100% of the other three vaccine strains. Analytical sensitivity of the Luminex assay was one to two logs lower than that of the reverse transcription real‐time PCR assay. Evaluated with 417 PRRSV‐2 positive clinical samples, 95% were detected by the Luminex assay. Compared to ORF5 sequencing results, the Luminex assay detected 92.4% (73/79) of MLV strains, 78.3% (18/23) of Fostera strains and 50% (2/4) of ATP strains. None of the 472 samples were the Prime Pac strain tested by either ORF5 sequencing or the Luminex assay. [ABSTRACT FROM AUTHOR]

Probabilistic solar irradiance forecasting has received widespread attention in recent years, as it provides more uncertainty information for the future photovoltaic generation. In this study, a hybrid probabilistic solar irradiance prediction method is proposed, which combines a deep recurrent neural network and residual modeling. Specifically, the long short‐term memory‐based point prediction using historical records and related features is applied to obtain deterministic forecasts. Next, these deterministic forecasts are employed as inputs to estimate the residual distributions. Furthermore, maximum likelihood estimation is utilized to compute the parameters of the residual distribution. Finally, the point prediction and residual distribution jointly generate the final probabilistic forecasting results. Compared with other deterministic and probabilistic forecasting models, the proposed method yields promising results on a publicly available dataset. © 2020 Institute of Electrical Engineers of Japan. Published by Wiley Periodicals LLC. [ABSTRACT FROM AUTHOR]

In recent years, reports indicated that PCV3 may be involved in porcine dermatitis and nephropathy syndrome (PDNS)‐like disease similar to that linked to PCV2. A total of 2,125 porcine samples from 910 cases were collected during 2016–2018 and tested for presence of PCV3 and PCV2 by real‐time PCR assays. Results showed high prevalence of PCV3 and PCV2: 28.4% samples from 41.2% cases were PCV3 positive and 16.4% samples from 16.7% cases were PCV2 positive. The overall coinfection rate was 5.4% and 8.4% at the sample and case level, respectively. Temporal analysis indicated that PCV3 positive case rate increased from 31.6% in 2016, 40.9% in 2017, to 55.6% in 2018. Although its prevalence was lower, PCV2‐positive case rate in 2018 (28.8%) doubled that in 2017 (14.4%). The coinfection case rate also increased from 3.4% in 2016, 8.0% in 2017 to 16.1% in 2018. The high positive rate of PCV3 (56.9%) and PCV2 (33.8%) in oral fluids, PCV3 in foetuses (57.1%) and PCV2 in tonsils (54.8%) implied viral transmission route and tissue tropism. In phylogenetic analysis, two small PCV3 clusters (1 and 2) were separated but others were clustered with low bootstrapping values indicating overall low genetic diversity. Genotypes, PCV2a‐h, were confirmed by analysing 2,944 strains, with a new genotype proposed as PCV2i. In this study, 61 PCV3 unique whole genomes were sequenced; 12 belonged to a separate cluster that were characterized by five consistent amino acid changes in the capsid protein (24V, 27K, 56D, 98R and 168K) and may be associated with potential differences in immunogenicity. Among the 43 unique PCV2 whole genomes sequenced, 31 belonged to PCV2d, 7 to PCV2a and 5 to PCV2b. Thus, our study demonstrates that PCV2d is the predominant genotype and PCV3 is widely circulating in the Midwest of the USA. [ABSTRACT FROM AUTHOR]

We identified influenza C virus (ICV) in samples from US cattle with bovine respiratory disease through real-time PCR testing and sequencing. Bovine ICV isolates had high nucleotide identities (≈98%) with each other and were closely related to human ICV strains (≈95%). Further research is needed to determine bovine ICV's zoonotic potential. [ABSTRACT FROM AUTHOR]

Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease resulting from abnormal interactions between T and B cells. The acquisition of SLE is linked to genetic susceptibility, and diverse environmental agents can trigger disease onset in genetically susceptible individuals. However, the strongest risk factor for developing SLE is being female (9:1 female to male ratio). The female sex steroid, estradiol, working through its receptors, contributes to the gender bias in SLE although the mechanisms remain enigmatic. In a small clinical trial, monthly administration of the estrogen receptor (ERα) antagonist, ICI182,780 (fulvestrant), significantly reduced disease indicators in SLE patients. In order to identify changes that could account for improved disease status, the present study utilized fulvestrant (Faslodex) to block ERα action in cultured SLE T cells that were purified from blood samples collected from SLE patients (n = 18, median age 42 years) and healthy control females (n = 25, median age 46 years). The effects of ERα antagonism on estradiol-dependent gene expression and canonical signaling pathways were analyzed. Pathways that were significantly altered by addition of Faslodex included T helper (Th) cell differentiation, steroid receptor signaling [glucocorticoid receptor (GR), ESR1 (ERα)], ubiquitination, and sumoylation pathways. ERα protein expression was significantly lower (p < 0.018) in freshly isolated, resting SLE T cells suggesting ERα turnover is inherently faster in SLE T cells. In contrast, ERα/ERβ mRNA and ERβ protein levels were not significantly different between SLE and normal control T cell samples. Plasma estradiol levels did not differ (p > 0.05) between SLE patients and controls. A previously undetected interaction between GR and ERα signaling pathways suggests posttranslational modification of steroid receptors in SLE T cells may alter ERα/GR actions and contribute to the strong gender bias of this autoimmune disorder. [ABSTRACT FROM AUTHOR]

Viruses belonging to the genus Bocaparvovirus(BoV) are a genetically diverse group of DNA viruses known to cause respiratory, enteric, and neurological diseases in animals, including humans. An intestinal sample from an alpaca (Vicugna pacos) herd with reoccurring diarrhea and respiratory disease was submitted for next-generation sequencing, revealing the presence of a BoV strain. The alpaca BoV strain (AlBoV) had a 58.58% whole genome nucleotide percent identity to a camel BoV from Dubai, belonging to a tentative ungulate BoV 8 species (UBoV8). Recombination events were lacking with other UBoV strains. The AlBoV genome was comprised of the NS1, NP1, and VP1 proteins. The NS1 protein had the highest amino acid percent identity range (57.89–67.85%) to the members of UBoV8, which was below the 85% cut-off set by the International Committee on Taxonomy of Viruses. The low NS1 amino acid identity suggests that AlBoV is a tentative new species. The whole genome, NS1, NP1, and VP1 phylogenetic trees illustrated distinct branching of AlBoV, sharing a common ancestor with UBoV8. Walker loop and Phospholipase A2 (PLA2) motifs that are vital for virus infectivity were identified in NS1 and VP1 proteins, respectively. Our study reports a novel BoV strain in an alpaca intestinal sample and highlights the need for additional BoV research. [ABSTRACT FROM AUTHOR]

Influenza is a common and contagious respiratory disease caused by influenza A, B, C, and D viruses (IAV, IBV, ICV, and IDV). A multiplex real-time RT-PCR assay was developed for simultaneous detection of IAV, IBV, ICV, and IDV. The assay was designed to target unique sequences in the matrix gene of IBV and ICV, the RNA polymerase subunit PB1 of IDV, and combined with USDA and CDC IAV assays, both target the matrix gene. The host 18S rRNA gene was included as an internal control. In silico analyses indicated high strain coverages: 97.9% for IBV, 99.5% for ICV, and 100% for IDV. Transcribed RNA, viral isolates and clinical samples were used for validation. The assay specifically detected target viruses without cross-reactivity, nor detection of other common pathogens. The limit of detection was approximately 30 copies for each viral RNA template, which was equivalent to a threshold cycle value of ~37. [ABSTRACT FROM AUTHOR]

Canine infectious respiratory disease (CIRD) is a complicated respiratory syndrome in dogs [1], [2], [3]. A panel PCR was developed [4] to detect nine pathogens commonly associated with CIRD: Mycoplasma cynos, Mycoplasma canis, Bordetella bronchiseptica; canine adenovirus type 2, canine herpesvirus 1, canine parainfluenza virus, canine distemper virus, canine influenza virus and canine respiratory coronavirus [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16]. To evaluate diagnostic performance of the assay, 740 nasal swab and lung tissue samples were collected and tested with the new assay, and compared to an older version of the assay detecting the same pathogens except that it does not differentiate the two Mycoplasma species. Results indicated that the new assay had the same level of specificity, but with higher diagnostic sensitivity and had identified additional samples with potential co-infections. To confirm the new assay is detecting the correct pathogens, samples with discrepant results between the two assays were sequence-confirmed. Spiking a high concertation target to samples carrying lower concentrations of other targets was carried out and the results demonstrated that there was no apparent interference among targets in the same PCR reaction. Another spike-in experiment was used to determine detection sensitivity between nasal swab and lung tissue samples, and similar results were obtained.•A nine-pathogen CIRD PCR panel assay had identified 139 positives from 740 clinical samples with 60 co-infections;•High-concentration target does not have apparent effect on detecting low-concentration targets;•Detection sensitivity were similar between nasal swab and lung tissue samples., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Author(s).)

Rotavirus C (RVC) is associated with acute diarrhoea in both children and young animals. Because of its frequent occurrence, additional sequences have recently been generated. In this study, we sequenced 21 complete genomes from porcine diarrhoea samples and analysed them together with all available reference sequences collected from the GenBank database [National Center for Biotechnology Information (NCBI)]. Based on phylogenetic analysis and genetic distance calculation, the number of each segment was identified as 31G, 26P, 13I, 5R, 5C, 5M, 12A, 10 N, 9T, 8E and 4 H for genotypes encoding VP7, VP4, VP6, VP1, VP2, VP3 and NSP1, NSP2, NSP3, NSP4 and NSP5, respectively. From the analysis, genotypes G19-G31, P[22]-P[26], R5, A9-A12, N9-N10, T7-T9 and E6-E8 were defined as newly identified genotypes, and genotype C6 was combined with C5, and M6 was combined with M1, due to their closely related nature. Estimated with the identity frequency ratio between the intergenotype and intragenotype, the nucleotide identity cutoff values for different genotypes were determined as 85, 85, 86, 84, 83, 84, 82, 87, 84, 81 and 79 % for VP7, VP4, VP6, VP1, VP2, VP3, NSP1, NSP2, NSP3, NSP4 and NSP5, respectively. Genotyping of the 49 US strains indicated possible segment reassortment in 9 of the 11 segments, with the exceptions being VP1 and NSP5, and the most prevalent genotypes for each segment genes in the USA were G6/G5/G21/G9-P5/P4-I6/I5-R1-C5-M1-A8-N1/N10-T1-E1-H1. Our study updated the genotypes of RVC strains and provided more evidence of RVC strain diversity that may be relevant to better understand genetic diversity, and the distribution and evolution of RVC strains.

Escherichia coli serogroups O157, O26, O45, O103, O111, O121, and O145, when carrying major virulence genes, the Shiga toxin genes stx 1 and stx 2 and the intimin gene eae , are important foodborne pathogens. They are referred to as the "top 7" Shiga toxin-producing E. coli (STEC) serogroups and were declared by the USDA as adulterants to human health. Since top 7 serogroup-positive cattle feces and ground beef can also contain nonadulterant E. coli strains, regular PCR cannot confirm whether the virulence genes are carried by adulterant or nonadulterant E. coli serogroups. Thus, traditional gold-standard STEC detection requires bacterial isolation and characterization, which are not compatible with high-throughput settings and often take a week to obtain a definitive result. In this study, we demonstrated that the partition-based multichannel digital PCR (dPCR) system can be used to detect and associate the E. coli serogroup-specific gene with major virulence genes and developed a single-cell-based dPCR approach for rapid (within 1 day) and accurate detection and confirmation of major STEC serogroups in high-throughput settings. Major virulence genes carried by each of the top 7 STEC serogroups were detected by dPCR with appropriately diluted intact bacterial cells from pure cultures, culture-spiked cattle feces, and culture-spiked ground beef. Furthermore, from 100 randomly collected, naturally shed cattle fecal samples, 3 O103 strains carrying eae and 2 O45 strains carrying stx 1 were identified by this dPCR assay and verified by the traditional isolation method. This novel and rapid dPCR assay is a culture-independent, high-throughput, accurate, and sensitive method for STEC detection and confirmation., (Copyright © 2020 American Society for Microbiology.)

Zearalenone (ZEA) is a non-steroidal estrogen mycotoxin produced by several Gibberella and Fusarium species. Accumulating evidence has indicated that ZEA strongly stimulates cell proliferation. However the detailed molecular and cellular mechanisms of ZEA-mediated induction of cell proliferation have not yet been completely explained. The aim of this study was to detect the role of miRNAs in ZEA-mediated induction of cell proliferation. The effects of ZEA on cell proliferation were assessed using a cell counting kit assay and xCELLigence system. Micro-RNA sequencing was performed after treatment of TM3 cells with ZEA (0.01 μmol/L) for different time periods (0, 2, 6 and 18 h). Cell function and pathway analysis of the miRNA target genes were performed by Ingenuity Pathway Analysis (IPA). We found that ZEA promotes TM3 cell proliferation at low concentrations. miRNA sequenceing revealed 66 differentially expressed miRNAs in ZEA-treated cells in comparison to the untreated control ( p < 0.05). The miRNA sequencing indicated that compared to control group, there were 66 miRNAs significant change ( p < 0.05) in ZEA-treated groups. IPA analysis showed that the predicated miRNAs target gene involved in cell Bio-functions including cell cycle, growth and proliferation, and in signaling pathways including MAPK and RAS-RAF-MEK-ERK pathways. Results from flow cytometry and Western Blot analysis validated the predictions that ZEA can affect cell cycle, and the MAPK signaling pathway. Taking these together, the cell proliferation induced ZEA is regulated by miRNAs. The results shed light on the molecular and cellular mechanisms for the mediation of ZEA to induce proliferation., Competing Interests: The authors declare no conflict of interest.

Influenza C virus (ICV) has been identified for the first time from bovine respiratory disease complex (BRDC) samples in the United States. Here, we report the complete genome sequence of the strain C/bovine/Montana/12/2016, identified from a nasal swab sample collected from a sick calf with clinical signs of respiratory disease in Montana.

Differentiation of Brucella canis from other Brucella species are mainly performed through PCR-based methods and multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) procedures. Both PCR-based and MLVA methods are limited in discriminating B. canis strains. A new MLVA-13Bc method for B. canis genotyping was established by combining eight newly-developed VNTRs with five published ones. During 2010 and 2016, 377 B. canis PCR-positives were identified from 6,844 canine blood samples from 22 U.S. states, resulting in 229 B. canis isolates. The MLVA-13Bc method was able to differentiate each of these 229 isolates. The Hunter-Gaston Discriminatory Index of the individual VNTR loci ranged from 0.516 to 0.934 and the combined discriminatory index reached 1.000. Three major clusters (A, B and C) and 10 genotype groups were identified from the 229 B. canis isolates. Cluster A mainly contains genotype groups 1 and 2, and a few group 3 isolates; nearly all Cluster B isolates were from group 6; other genotype groups were classified into Cluster C. Our newly developed MLVA-13Bc assay is a highly discriminatory assay for B. canis genotyping, and can serve as a useful molecular epidemiological tool, especially for tracing the source of contamination in an event of a B. canis outbreak.

A microarray developed on the basis of 2895 unique transcripts from larval gut was used to compare gut gene expression profiles between a laboratory-selected Cry1Ab-resistant (R) strain and its isoline susceptible (S) strain of the European corn borer (Ostrinia nubilalis) after the larvae were fed the leaves of transgenic corn (MON810) expressing Cry1Ab or its non-transgenic isoline for 6 h. We revealed 398 gut genes differentially expressed (i.e., either up- or down-regulated genes with expression ratio ≥2.0) in S-strain, but only 264 gut genes differentially expressed in R-strain after being fed transgenic corn leaves. Although the percentages of down-regulated genes among the total number of differentially expressed genes (50% in S-strain and 45% in R-strain) were similar between the R- and S-strains, the expression ratios of down-regulated genes were much higher in S-strain than in R-strain. We revealed that 17 and 9 significantly up- or down-regulated gut genes from S and R-strain, respectively, including serine proteases and aminopeptidases. These genes may be associated with Cry1Ab toxicity by degradation, binding, and cellular defense. Overall, our study suggests enhanced adaptation of Cry1Ab-resistant larvae on transgenic Cry1Ab corn as revealed by lower number and lower ratios of differentially expressed genes in R-strain than in S-strain of O. nubilalis.

Background: Genome assembly remains an unsolved problem. Assembly projects face a range of hurdles that confound assembly. Thus a variety of tools and approaches are needed to improve draft genomes., Results: We used a custom assembly workflow to optimize consensus genome map assembly, resulting in an assembly equal to the estimated length of the Tribolium castaneum genome and with an N50 of more than 1 Mb. We used this map for super scaffolding the T. castaneum sequence assembly, more than tripling its N50 with the program Stitch., Conclusions: In this article we present software that leverages consensus genome maps assembled from extremely long single molecule maps to increase the contiguity of sequence assemblies. We report the results of applying these tools to validate and improve a 7x Sanger draft of the T. castaneum genome.

We developed a microarray based on 2895 unique transcripts assembled from 15,000 cDNA sequences from the European corn borer (Ostrinia nubilalis) larval gut. This microarray was used to monitor gene expression in early third-instar larvae of Bacillus thuringiensis (Bt)-susceptible O. nubilalis after 6 h feeding on diet, with or without the Bt Cry1Ab protoxin. We identified 174 transcripts, for which the expression was changed more than two-fold in the gut of the larvae fed Cry1Ab protoxin (p < 0.05), representing 80 down-regulated and 94 up-regulated transcripts. Among 174 differentially expressed transcripts, 13 transcripts putatively encode proteins that are potentially involved in Bt toxicity, and these transcripts include eight serine proteases, three aminopeptidases, one alkaline phosphatase, and one cadherin. The expressions of trypsin-like protease and three aminopeptidase transcripts were variable, but two potential Bt-binding proteins, alkaline phosphatase and cadherin were consistently up-regulated in larvae fed Cry1Ab protoxin. The significantly up and down-regulated transcripts may be involved in Cry1Ab toxicity by activation, degradation, toxin binding, and other related cellular responses. This study is a preliminary survey of Cry1Ab protoxin-induced transcriptional responses in O. nubilalis gut and our results are expected to help with further studies on Bt toxin-insect interactions at the molecular level.

The insect cuticle is a composite biomaterial made up primarily of chitin and proteins. The physical properties of the cuticle can vary greatly from hard and rigid to soft and flexible. Understanding how different cuticle types are assembled can aid in the development of novel biomimetic materials for use in medicine and technology. Toward this goal, we have taken a combined proteomics and transcriptomics approach with the red flour beetle, Tribolium castaneum, to examine the protein and gene expression profiles of the elytra and hindwings, appendages that contain rigid and soft cuticles, respectively. Two-dimensional gel electrophoresis analysis revealed distinct differences in the protein profiles between elytra and hindwings, with four highly abundant proteins dominating the elytral cuticle extract. MALDI/TOF mass spectrometry identified 19 proteins homologous to known or hypothesized cuticular proteins (CPs), including a novel low complexity protein enriched in charged residues. Microarray analysis identified 372 genes with a 10-fold or greater difference in transcript levels between elytra and hindwings. CP genes with higher expression in the elytra belonged to the Rebers and Riddiford family (CPR) type 2, or cuticular proteins of low complexity (CPLC) enriched in glycine or proline. In contrast, a majority of the CP genes with higher expression in hindwings were classified as CPR type 1, cuticular proteins analogous to peritrophins (CPAP), or members of the Tweedle family. This research shows that the elyra and hindwings, representatives of rigid and soft cuticles, have different protein and gene expression profiles for structural proteins that may influence the mechanical properties of these cuticles.

Exercise has been linked to a reduced cancer risk in animal models. However, the underlying mechanisms are unclear. This study assessed the effect of exercise with dietary consideration on the phospholipid profile in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse skin tissues. CD-1 mice were randomly assigned to one of the three groups: ad libitum-fed sedentary control; ad libitum-fed treadmill exercise at 13.4 m/min for 60 min/d, 5 d/wk (Ex+AL); and treadmill-exercised but pair-fed with the same amount as the control (Ex+PF). After 14 weeks, Ex+PF but not Ex+AL mice showed approximately 25% decrease in both body weight and body fat when compared with the controls. Of the total 338 phospholipids determined by electrospray ionization-tandem mass spectrometry, 57 were significantly changed, and 25 species could distinguish effects of exercise and diet treatments in a stepwise discriminant analysis. A 36% to 75% decrease of phosphatidylinositol (PI) levels in Ex+PF mice occurred along with a significant reduction of PI 3-kinase in TPA-induced skin epidermis, as measured by both Western blotting and immunohistochemistry. In addition, approximately 2-fold increase of the long-chain polyunsaturated fatty acids, docosahexaenoic and docosapentaenoic acids, in phosphatidylcholines, phosphatidylethanolamines, and lysophosphatidylethanolamines was observed in the Ex+PF group. Microarray analysis indicated that the expression of fatty acid elongase-1 increased. Taken together, these data indicate that exercise with controlled dietary intake, but not exercise alone, significantly reduced body weight and body fat as well as modified the phospholipid profile, which may contribute to cancer prevention by reducing TPA-induced PI 3-kinase and by enhancing omega-3 fatty acid elongation., ((c) 2010 AACR.)