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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.

Infectious bovine keratoconjunctivitis (IBK), also known as pinkeye, is one of the most common eye diseases in cattle. Several pathogens have been associated with IBK cases, however, Moraxella bovis, Moraxella bovoculi, Mycoplasma bovis, Mycoplasma bovoculi and bovine herpesvirus type 1 (BHV-1) are most frequently observed. A multiplex real-time PCR assay using two reactions was developed for the detection and differentiation of these five pathogens. Detection sensitivities of the multiplex assays were compared to singleplex reactions testing for the same targets. Correlation coefficients (R2) of >0.99, and PCR efficiencies between 92 and 106% were demonstrated in all singleplex and multiplex real-time PCR reactions. The limits of detection (LOD) of multiplex assays for Moraxella bovis, Moraxella bovoculi, Mycoplasma bovis, Mycoplasma bovoculi and BHV-1 were 19, 23, 25, 24 and 26 copies per reaction, respectively. No cross amplification was observed for specificity testing of 179 IBK positive clinical samples and 55 non-target clinical samples. Percentage of clinical samples positive for Mycoplasma bovoculi, Moraxella bovoculi, Moraxella bovis, BHV-1 and Mycoplasma bovis were 88.8% (159/179), 75.9% (136/179), 60.3% (108/179), 11.7% (21/179) and 10.0% (18/179), respectively. Moraxella bovis, Moraxella bovoculi and Mycoplasma bovoculi were more prevalent than Mycoplasma bovis and BHV-1 in IBK samples collected from animals in this study population. Our data indicates that the multiplex real-time PCR panel assay is highly sensitive and highly specific for the detection and differentiation of the five major pathogens associated with bovine pinkeye., Highlights • A multiplex real-time PCR is developed for the detection of five major IBK pathogens. • Correlation coefficients of all standard curves were >0.99. • PCR amplification efficiencies for the five pathogens were between 92% and 106%. • Limits of detection were between 19 and 26 copies per reaction for the five pathogens. • Moraxella bovoculi, Mycoplasma bovoculi, and Moraxella bovis were more prevalent IBK pathogens.

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.

The lower Dipteran fungus fly,Sciara coprophila, has many unique biological features. For example,Sciaraundergoes paternal chromosome elimination and maternal X chromosome nondisjunction during spermatogenesis, paternal X elimination during embryogenesis, intrachromosomal DNA amplification of DNA puff loci during larval development, and germline-limited chromosome elimination from all somatic cells. Paternal chromosome elimination inSciarawas the first observation of imprinting, though the mechanism remains a mystery. Here, we present the first draft genome sequence forSciara coprophilato take a large step forward in aiding these studies. We approached assembling theSciaragenome using multiple sequencing technologies: PacBio, Oxford Nanopore MinION, and Illumina. To find an optimal assembly using these datasets, we generated 44 Illumina assemblies using 7 short-read assemblers and 50 long-read assemblies of PacBio and MinION sequence data using 6 long-read assemblers. We ranked assemblies using a battery of reference-free metrics, and scaffolded a subset of the highest-ranking assemblies using BioNano Genomics optical maps. RNA-seq datasets from multiple life stages and both sexes facilitated genome annotation. Moreover, we anchored nearly half of theSciaragenome sequence into chromosomes. Finally, we used the signal level of both the PacBio and Oxford Nanopore data to explore the presence or absence of DNA modifications in theSciaragenome since DNA modifications may play a role in imprinting inSciara, as they do in mammals. These data serve as the foundation for future research by the growing community studying the unique features of this emerging model system.

Streptococcus equi subsp. equi is a Gram positive bacterial pathogen commonly associated with strangles in horses, a respiratory disease characterized by abscessation of submandibular and retropharyngeal lymph nodes which can lead to obstruction of the airway. Several real-time PCR (qPCR) assays have been developed for detection of S. equi from horses with many targeting conserved regions of the S. equi cell wall-associated M-protein (SeM), a major virulence factor and immunogen of S. equi. Our objective was to develop a nested PCR (nPCR) targeting SeM and an 18S rRNA internal control gene for detection of S. equi from horses with potential improvement in detection sensitivity compared to a qPCR. Primers and probes from the Kansas State Veterinary Diagnostic Laboratory (KSVDL) S. equi clinical testing assay were utilized for all qPCR testing. Primers flanking the SeM qPCR target region were selected for an initial end-point PCR step of the nested assay; PCR product from the end-point reaction then served as template for the qPCR reaction step of the nested assay. Sample nucleic acid was also tested directly with qPCR to allow for assay comparison. Nucleic acid from clinical specimens (n = 188) submitted to KSVDL were tested in parallel with each assay. The nPCR and qPCR assays identified 22.9% (43/188) and 13.3% (25/188) of samples positive for S. equi, respectively. None of the samples positive by qPCR were negative by nPCR. The PCR products from all positive samples were submitted for DNA sequencing. Each of the 25 samples positive by both assays had a high nucleotide identity match (>96%) to the SeM gene. Among the samples positive by nPCR but negative by qPCR, 17 of 18 were sequence confirmed for SeM at greater than 96% nucleotide identity. Based on the nPCR Ct (37.8) of the one sequence un-confirmed case, it is likely that the S. equi bacterial load in this sample was below the necessary concentration for successful sequencing. Limit of detection (LOD) for the nPCR was established at a Ct of 37, and based both on the LOD of the qPCR assay (Ct of 37), as determined by standard curve data, and on the highest nPCR Cts (~37) of clinical samples able to result in SeM sequence-confirmation. As demonstrated by sequencing confirmation, the nPCR assay targeting the SeM gene is highly specific to S. equi. The increased sensitivity of the nPCR, compared to the qPCR, may reduce the number of false negative sample results in clinical testing and provide a superior detection method during low bacterial shedding periods.

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.

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.

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 &mu, mol/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 &lt, 0.05). The miRNA sequencing indicated that compared to control group, there were 66 miRNAs significant change (p &lt, 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.

Rice (Oryza sativa L.) is not susceptible to rust fungi, including the wheat leaf rust fungus Puccinia triticina. Upon inoculation with P. triticina spores, infection hyphae and appressoria were observed on the leaf surfaces of the rice cultivar Nipponbare. The cultivar responded to the inoculation with brown discoloration of the local tissue and fragmentation of rust infection hyphae and appressoria. A microarray gene-expression analysis of the host transcriptional response was performed 24 h after inoculation, revealing rice genes that were up- or down-regulated following the interaction. In particular, the loci represented by five probe sets (Os.55776.1. S1_x_at, Os.55647.1. A1_at, Os.55776.1. S1_at, OsAffx.10944.1. S1_x_at, and OsAffx.10944.1. S1_at) displayed the highest increase in gene expression compared to the control inoculation. The probe sets included members of the receptor-like kinase family (RLK) that occurs within a cluster of RLK genes on chromosome 1. Other RLK genes, within the RLK gene cluster and at another location, also showed increases in gene expression after P. triticina inoculation. The RLK genes varied in response to challenges with different rust strains or when challenged with several non-rust wheat pathogens that are also non-pathogenic to rice. The results indicate that rice has an active transcriptional and possible defense priming reaction in response to P. triticina and other non-host fungal pathogens.

Zearalenone (ZEA), a non-steroidal estrogen mycotoxin produced by several species of Fusarium fungi, can be metabolized into many other derivatives by microorganisms, plants, animals and humans. It can affect mammalian reproductive capability by impacting the synthesis and secretion of sex hormones, including testosterone, estradiol and progesterone. This review summarizes the mechanisms in which ZEA and its derivatives disturb the synthesis and secretion of sex steroid hormones. Because of its structural analogy to estrogen, ZEA and its derivatives can exert a variety of estrogen-like effects and engage in estrogen negative feedback regulation, which can result in mediating the production of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the pituitary gland. ZEA and its derivatives can ultimately reduce the number of Leydig cells and granulosa cells by inducing oxidative stress, endoplasmic reticulum (ER) stress, cell cycle arrest, cell apoptosis, and cell regeneration delay. Additionally, they can disrupt the mitochondrial structure and influence mitochondrial functions through overproduction of reactive oxygen species (ROS) and aberrant autophagy signaling ways. Finally, ZEA and its derivatives can disturb the expressions and activities of the related steroidogenic enzymes through cross talking between membrane and nuclear estrogen receptors.

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.

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 0.05) 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.

We developed a high-resolution expression microarray based on 2456 unique transcripts from a cDNA library of the aquatic midge (Chironomus tentans). By using the microarray, we detected that 146, 434 and 243 genes were differentially expressed after C. tentans larvae were exposed to chlorpyrifos (organophosphate insecticide) at 0.1 and 0.5 μg/L, and atrazine (triazine herbicide) at 1000 μg/L, respectively, for 48 h. The number of differentially expressed genes in the larvae exposed to chlorpyrifos at 0.5 μg/L was three times of that in the larvae exposed to chlorpyrifos at 0.1 μg/L. Among the differentially expressed genes in response to chlorpyrifos exposures, 76 genes showed significant Blast hits, and among them 42 were in common between the chlorpyrifos and atrazine exposures. In 19 differentially expressed xenobiotic detoxification genes, 16 were significantly up-regulated in the larvae exposed to chlorpyrifos and/or atrazine. Two cytochrome P450 genes (CtCYP6EV1 and CtCYP4DG2) were specifically up-regulated by chlorpyrifos, whereas three cytochrome P450 genes (CtCYP4DG1, CtCYP6EX3 and CtCYP6EV3) were specifically up-regulated by atrazine. Our results showed that chlorpyrifos exposures even at low concentrations can lead to significant changes in gene expression. The significant transcriptional responses are likely attributed to larval intoxication by the insecticide. These results not only support our previous studies in which candidate gene approaches were used, but also can potentially help develop specific molecular markers for monitoring pesticide exposures in non-target organisms in aquatic systems.

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.

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.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1911-8) contains supplementary material, which is available to authorized users.