Whole-genome classification of rotavirus C and genetic diversity of porcine strains in the USA.

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.

High-resolution melting curve FRET-PCR rapidly identifies SARS-CoV-2 mutations.

Reverse transcription fluorescence resonance energy transfer-polymerase chain reaction (FRET-PCRs) were designed against the two most common mutations in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) (A23403G in the spike protein; C14408T in the RNA-dependent RNA polymerase). Based on high-resolution melting curve analysis, the reverse transcription (RT) FRET-PCRs identified the mutations in american type culture collection control viruses, and feline and human clinical samples. All major makes of PCR machines can perform melting curve analysis and thus further specifically designed FRET-PCRs could enable active surveillance for mutations and variants in countries where genome sequencing is not readily available.

Single-Cell-Based Digital PCR Detection and Association of Shiga Toxin-Producing Escherichia coli Serogroups and Major Virulence Genes.

Escherichia coli serogroups O157, O26, O45, O103, O111, O121, and O145, when carrying major virulence genes, the Shiga toxin genes stx1 and stx2 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 stx1 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.

Polymerase Chain Reaction-Based Prevalence of Serogroups of Escherichia coli Known to Carry Shiga Toxin Genes in Feces of Finisher Pigs.

Shiga toxin-producing Escherichia coli (STEC) are major foodborne pathogens and seven serogroups, O26, O45, O103, O111, O121, O145, and O157, that account for the majority of the STEC-associated illness in humans. Similar to cattle, swine also harbor STEC and shed them in the feces and can be a source of human STEC infections. Information on the prevalence of STEC in swine feces is limited. Therefore, our objective was to utilize polymerase chain reaction (PCR) assays to determine prevalence of major virulence genes and serogroups of STEC. Fecal samples (n = 598), collected from finisher pigs within 3 weeks before marketing in 10 pig flows located in 8 states, were included in the study. Samples enriched in E. coli broth were subjected to a real-time PCR assay targeting three virulence genes, Shiga toxin 1 (stx1), Shiga toxin 2 (stx2), and intimin (eae), which encode for Shiga toxins 1 and 2, and intimin, respectively. A novel PCR assay was designed and validated to detect serogroups, O8, O20, O59, O86, O91, O100, O120, and O174, previously reported to be commonly present in swine feces. In addition, enriched fecal samples positive for Shiga toxin genes were subjected to a multiplex PCR assay targeting O26, O45, O103, O104, O111, O121, O145, and O157 serogroups implicated in human clinical infections. Of the 598 fecal samples tested by real-time PCR, 25.9%, 65.1%, and 67% were positive for stx1, stx2, and eae, respectively. The novel eight-plex PCR assay indicated the predominant prevalence of O8 (88.6%), O86 (35.5%), O174 (24.1%), O100 (20.2%), and O91 (15.6%) serogroups. Among the seven serogroups relevant to human infections, three serogroups, O121 (17.6%), O157 (14%), and O26 (11%) were predominant. PCR-based detection indicated high prevalence of Shiga toxin genes and serogroups that are known to carry Shiga toxin genes, including serogroups commonly prevalent in cattle feces and implicated in human infections and in edema disease in swine.

Seasonal Presence of Salmonella spp., Salmonella Typhimurium and Its Monophasic Variant Serotype I 4,[5],12:i:-, in Selected United States Swine Feed Mills.

This study evaluated the seasonal prevalence and distribution of Salmonella spp., Salmonella enterica serovar Typhimurium (ST) and its monophasic variant 4,[5],12:i:- (STM), in selected swine feed mills across the United States. Eleven facilities were selected for this study and 12 sites were sampled within each mill during fall 2016, early spring 2017, and summer 2017. Samples were evaluated following the USDA-FSIS guidelines for Salmonella isolation and culture positive samples were analyzed by polymerase chain reaction (PCR). A multiplex real-time PCR was used to differentiate ST and STM from other serotypes. Associations between season, mill, and sample site with Salmonella presence were investigated using generalized linear mixed models. Both season (p < 0.007) and mill (p < 0.005) were significantly associated with Salmonella spp. presence. Fall months were associated with a higher Salmonella prevalence (13.2%) compared with early spring and summer. A total of five isolates, among the 383 samples were serotyped as ST and STM. These two serotypes showed a similar seasonal presence throughout the study, being found during fall and summer seasons. These findings demonstrated the seasonal presence of Salmonella spp. in feed mills and the role of these environments as potential pathogen entry route into the human food chain.

Influenza C Virus in Cattle with Respiratory Disease, United States, 2016-2018.

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.

Mutations in a Highly Conserved Motif of nsp1ß Protein Attenuate the Innate Immune Suppression Function of Porcine Reproductive and Respiratory Syndrome Virus.

UNLABELLED: Porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 1ß (nsp1ß) is a multifunctional viral protein, which is involved in suppressing the host innate immune response and activating a unique -2/-1 programmed ribosomal frameshifting (PRF) signal for the expression of frameshifting products. In this study, site-directed mutagenesis analysis showed that the R128A or R129A mutation introduced into a highly conserved motif ((123)GKYLQRRLQ(131)) reduced the ability of nsp1ß to suppress interferon beta (IFN-ß) activation and also impaired nsp1ß's function as a PRF transactivator. Three recombinant viruses, vR128A, vR129A, and vRR129AA, carrying single or double mutations in the GKYLQRRLQ motif were characterized. In comparison to the wild-type (WT) virus, vR128A and vR129A showed slightly reduced growth abilities, while the vRR129AA mutant had a significantly reduced growth ability in infected cells. Consistent with the attenuated growth phenotype in vitro, pigs infected with nsp1ß mutants had lower levels of viremia than did WT virus-infected pigs. Compared to the WT virus in infected cells, all three mutated viruses stimulated high levels of IFN-α expression and exhibited a reduced ability to suppress the mRNA expression of selected interferon-stimulated genes (ISGs). In pigs infected with nsp1ß mutants, IFN-α production was increased in the lungs at early time points postinfection, which was correlated with increased innate NK cell function. Furthermore, the augmented innate response was consistent with the increased production of IFN-γ in pigs infected with mutated viruses. These data demonstrate that residues R128 and R129 are critical for nsp1ß function and that modifying these key residues in the GKYLQRRLQ motif attenuates virus growth ability and improves the innate and adaptive immune responses in infected animals. IMPORTANCE: PRRSV infection induces poor antiviral innate IFN and cytokine responses, which results in weak adaptive immunity. One of the strategies in next-generation vaccine construction is to manipulate viral proteins/genetic elements involved in antagonizing the host immune response. PRRSV nsp1ß was identified to be a strong innate immune antagonist. In this study, two basic amino acids, R128 and R129, in a highly conserved GKYLQRRLQ motif were determined to be critical for nsp1ß function. Mutations introduced into these two residues attenuated virus growth and improved the innate and adaptive immune responses of infected animals. Technologies developed in this study could be broadly applied to current commercial PRRSV modified live-virus (MLV) vaccines and other candidate vaccines.

Pathogenicity and transmissibility of novel reassortant H3N2 influenza viruses with 2009 pandemic H1N1 genes in pigs.

UNLABELLED: At least 10 different genotypes of novel reassortant H3N2 influenza viruses with 2009 pandemic H1N1 [A(H1N1)pdm09] gene(s) have been identified in U.S. pigs, including the H3N2 variant with a single A(H1N1)pdm09 M gene, which has infected more than 300 people. To date, only three genotypes of these viruses have been evaluated in animal models, and the pathogenicity and transmissibility of the other seven genotype viruses remain unknown. Here, we show that three H3N2 reassortant viruses that contain 3 (NP, M, and NS) or 5 (PA, PB2, NP, M, and NS) genes from A(H1N1)pdm09 were pathogenic in pigs, similar to the endemic H3N2 swine virus. However, the reassortant H3N2 virus with 3 A(H1N1)pdm09 genes and a recent human influenza virus N2 gene was transmitted most efficiently among pigs, whereas the reassortant H3N2 virus with 5 A(H1N1)pdm09 genes was transmitted less efficiently than the endemic H3N2 virus. Interestingly, the polymerase complex of reassortant H3N2 virus with 5 A(H1N1)pdm09 genes showed significantly higher polymerase activity than those of endemic and reassortant H3N2 viruses with 3 A(H1N1)pdm09 genes. Further studies showed that an avian-like glycine at position 228 at the hemagglutinin (HA) receptor binding site is responsible for inefficient transmission of the reassortant H3N2 virus with 5 A(H1N1)pdm09 genes. Taken together, our results provide insights into the pathogenicity and transmissibility of novel reassortant H3N2 viruses in pigs and suggest that a mammalian-like serine at position 228 in the HA is critical for the transmissibility of these reassortant H3N2 viruses. IMPORTANCE: Swine influenza is a highly contagious zoonotic disease that threatens animal and public health. Introduction of 2009 pandemic H1N1 virus [A(H1N1)pdm09] into swine herds has resulted in novel reassortant influenza viruses in swine, including H3N2 and H1N2 variants that have caused human infections in the United States. We showed that reassortant H3N2 influenza viruses with 3 or 5 genes from A(H1N1)pdm09 isolated from diseased pigs are pathogenic and transmissible in pigs, but the reassortant H3N2 virus with 5 A(H1N1)pdm09 genes displayed less efficient transmissibility than the endemic and reassortant H3N2 viruses with 3 A(H1N1)pdm09 genes. Further studies revealed that an avian-like glycine at the HA 228 receptor binding site of the reassortant H3N2 virus with 5 A(H1N1)pdm09 genes is responsible for less efficient transmissibility in pigs. Our results provide insights into viral pathogenesis and the transmission of novel reassortant H3N2 viruses that are circulating in U.S. swine herds and warrant future surveillance.

Pigs immunized with a novel E2 subunit vaccine are protected from subgenotype heterologous classical swine fever virus challenge.

BACKGROUND: Classical swine fever (CSF) or hog cholera is a highly contagious swine viral disease. CSF endemic countries have to use routine vaccination with modified live virus (MLV) vaccines to prevent and control CSF. However, it is impossible to serologically differentiate MLV vaccinated pigs from those infected with CSF virus (CSFV). The aim of this study is to develop a one-dose E2-subunit vaccine that can provide protection against CSFV challenge. We hypothesize that a vaccine consisting of a suitable adjuvant and recombinant E2 with natural conformation may induce a similar level of protection as the MLV vaccine. RESULTS: Our experimental vaccine KNB-E2 was formulated with the recombinant E2 protein (Genotype 1.1) expressed by insect cells and an oil-in-water emulsion based adjuvant. 10 pigs (3 weeks old, 5 pigs/group) were immunized intramuscularly with one dose or two doses (3 weeks apart) KNB-E2, and 10 more control pigs were administered normal saline solution only. Two weeks after the second vaccination, all KNB-E2 vaccinated pigs and 5 control pigs were challenged with 5 × 10(5) TCID50 CSFV Honduras/1997 (Genotype 1.3, 1 ml intramuscular, 1 ml intranasal). It was found that while control pigs infected with CSFV stopped growing and developed high fever (>40 °C), high level CSFV load in blood and nasal fluid, and severe leukopenia 3-14 days post challenge, all KNB-E2 vaccinated pigs continued to grow as control pigs without CSFV exposure, did not show any fever, had low or undetectable level of CSFV in blood and nasal fluid. At the time of CSFV challenge, only pigs immunized with KNB-E2 developed high levels of E2-specific antibodies and anti-CSFV neutralizing antibodies. CONCLUSIONS: Our studies provide direct evidence that pigs immunized with one dose KNB-E2 can be protected clinically from CSFV challenge. This protection is likely mediated by high levels of E2-specific and anti-CSFV neutralizing antibodies.

A Four-Plex Real-Time PCR Assay, Based on rfbE, stx1, stx2, and eae Genes, for the Detection and Quantification of Shiga Toxin-Producing Escherichia coli O157 in Cattle Feces.

Several real-time polymerase chain reaction (PCR) assays have been developed to detect and quantify Shiga toxin-producing Escherichia coli (STEC) O157:H7, but none have targeted the O-antigen specific gene (rfbEO157) in combination with the three major virulence genes, stx1, stx2, and eae. Our objectives were to develop and validate a four-plex, quantitative PCR (mqPCR) assay targeting rfbE(O157), stx1, stx2, and eae for the detection and quantification of STEC O157 in cattle feces, and compare the applicability of the assay to detect STEC O157 to a culture method and conventional PCR (cPCR) targeting the same four genes. Specificity of the mqPCR assay to differentially detect the four genes was confirmed with strains of O157 and non-O157 STEC with different profiles of target genes. In cattle feces spiked with pure cultures, detection limits were 2.8×10(4) and 2.8×10(0) colony-forming units/g before and after enrichment, respectively. Detection of STEC O157 in feedlot cattle fecal samples (n=278) was compared between mqPCR, cPCR, and a culture method. The mqPCR detected 48.9% (136/278) of samples as positive for E. coli O157. Of the 100 samples that were randomly picked from 136 mqPCR-positive samples, 35 and 48 tested positive by cPCR and culture method, respectively. Of the 100 samples randomly chosen from 142 mqPCR-negative samples, all were negative by cPCR, but 21 samples tested positive by the culture method. McNemar's chi-square tests indicated significant disagreement between the proportions of positive samples detected by the three methods. In conclusion, the mqPCR assay that targets four genes is a novel and more sensitive method than the cPCR or culture method to detect STEC O157 in cattle feces. However, the use of real-time PCR as a screening method to identify positive samples and then subjecting only positive samples to a culture method may underestimate the presence of STEC O157 in fecal samples.

Escherichia coli O26 in feedlot cattle: fecal prevalence, isolation, characterization, and effects of an E. coli O157 vaccine and a direct-fed microbial.

Escherichia coli O26 is second only to O157 in causing foodborne, Shiga toxin-producing E. coli (STEC) infections. Our objectives were to determine fecal prevalence and characteristics of E. coli O26 in commercial feedlot cattle (17,148) that were enrolled in a study to evaluate an E. coli O157:H7 siderophore receptor and porin (SRP(®)) vaccine (VAC) and a direct-fed microbial (DFM; 10(6) colony-forming units [CFU]/animal/day of Lactobacillus acidophilus and 10(9) CFU/animal/day of Propionibacterium freudenreichii). Cattle were randomly allocated to 40 pens within 10 complete blocks; pens were randomly assigned to control, VAC, DFM, or VAC+DFM treatments. Vaccine was administered on days 0 and 21, and DFM was fed throughout the study. Pen-floor fecal samples (30/pen) were collected weekly for the last 4 study weeks. Samples were enriched in E. coli broth and subjected to a multiplex polymerase chain reaction (PCR) designed to detect O26-specific wzx gene and four major virulence genes (stx1, stx2, eae, and ehxA) and to a culture-based procedure that involved immunomagnetic separation and plating on MacConkey agar. Ten presumptive E. coli colonies were randomly picked, pooled, and tested by the multiplex PCR. Pooled colonies positive for O26 serogroup were streaked on sorbose MacConkey agar, and 10 randomly picked colonies per sample were tested individually by the multiplex PCR. The overall prevalence of E. coli O26 was higher (p<0.001) by the culture-based method compared to the PCR assay (22.7 versus 10.5%). The interventions (VAC and or DFM) had no impact on fecal shedding of O26. Serogroup O26 was recovered in pure culture from 23.9% (260 of 1089) of O26 PCR-positive pooled colonies. Only 7 of the 260 isolates were positive for the stx gene and 90.1% of the isolates possessed an eaeß gene that codes for intimin subtype ß, but not the bfpA gene, which codes for bundle-forming pilus. Therefore, the majority of the O26 recovered from feedlot cattle feces was atypical enteropathogenic E. coli, and not STEC.

Unlocking the Potential of Human-Induced Pluripotent Stem Cells: Cellular Responses and Secretome Profiles in Peptide Hydrogel 3D Culture.

Human-induced pluripotent stem cells (hiPSCs) have shown great potential for human health, but their growth and properties have been significantly limited by the traditional monolayer (2D) cell culture method for more than 15 years. Three-dimensional (3D) culture technology has demonstrated tremendous advantages over 2D. In particular, the 3D PGmatrix hiPSC derived from a peptide hydrogel offers a breakthrough pathway for the maintenance and expansion of physiologically relevant hiPSC 3D colonies (spheroids). In this study, the impact of 3D culture conditions in PGmatrix hiPSC on cell performance, integrity, and secretome profiles was determined across two commonly used hiPSC cell lines derived from fibroblast cells (hiPSC-F) and peripheral blood mononuclear cells (hiPSC-P) in the two most popular hiPSC culture media (mTeSR1 and essential eight (E8)). The 3D culture conditions varied in hydrogel strength, 3D embedded matrix, and 3D suspension matrix. The results showed that hiPSCs cultured in 3D PGmatrix hiPSC demonstrated the ability to maintain a consistently high cell viability that was above 95% across all the 3D conditions with cell expansion rates of 10-20-fold, depending on the 3D conditions and cell lines. The RT-qPCR analysis suggested that pluripotent gene markers are stable and not significantly affected by the cell lines or 3D PGmatrix conditions tested in this study. Mass spectrometry-based analysis of secretome from hiPSCs cultured in 3D PGmatrix hiPSC revealed a significantly higher quantity of unique proteins, including extracellular vesicle (EV)-related proteins and growth factors, compared to those in the 2D culture. Moreover, this is the first evidence to identify that hiPSCs in a medium with a rich supplement (i.e., mTeSR1) released more growth-regulating factors, while in a medium with fewer supplements (i.e., E8) hiPSCs secreted more survival growth factors and extracellular proteins. These findings offer insights into how these differences may impact hiPSC behavior, and they deepen our understanding of how hiPSCs respond to 3D culture conditions, aiding the optimization of hiPSC properties in translational biomedical research toward clinical applications.

Evaluation of Truck Cab Decontamination Procedures following Inoculation with Porcine Epidemic Diarrhea Virus and Porcine Reproductive and Respiratory Syndrome Virus.

This experiment aimed to evaluate commercially available disinfectants and their application methods against porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV) on truck cab surfaces. Plastic, fabric, and rubber surfaces inoculated with PEDV or PRRSV were placed in a full-scale truck cab and then treated with one of eight randomly assigned disinfectant treatments. After application, surfaces were environmentally sampled with cotton gauze and tested for PEDV and PRRSV using qPCR duplex analysis. There was a disinfectant × surface interaction (p < 0.0001), indicating a detectable amount of PEDV or PRRSV RNA was impacted by disinfectant treatment and surface material. For rubber surfaces, 10% bleach application had lower detectable amounts of RNA compared to all other treatments (p < 0.05) except Intervention via misting fumigation, which was intermediate. In both fabric and plastic surfaces, there was no evidence (p > 0.05) of a difference in detectable RNA between disinfectant treatments. For disinfectant treatments, fabric surfaces with no chemical treatment had less detectable viral RNA compared to the corresponding plastic and rubber (p < 0.05). Intervention applied via pump sprayer to fabric surfaces had less detectable viral RNA than plastic (p < 0.05). Furthermore, 10% bleach applied via pump sprayer to fabric and rubber surfaces had less detectable viral RNA than plastic (p < 0.05). Also, a 10 h downtime, with no chemical application or gaseous fumigation for 10 h, applied to fabric surfaces had less detectable viral RNA than other surfaces (p < 0.05). Sixteen treatments were evaluated via swine bioassay, but all samples failed to produce infectivity. In summary, commercially available disinfectants successfully reduced detectable viral RNA on surfaces but did not eliminate viral genetic material, highlighting the importance of bioexclusion of pathogens of interest.

Prevalence of Escherichia coli O-types and Shiga toxin genes in fecal samples from feedlot cattle.

While efforts to control foodborne illness associated with the Shiga toxin-producing Escherichia coli (E. coli) O157 through processes and procedures implemented at harvest facilities have been very successful, there is concern about the burden of illness associated with other Shiga toxin-producing E. coli. The U.S. Department of Agriculture Food Safety and Inspection Service announced plans to classify an additional six non-O157 Shiga toxin-producing E. coli as adulterants. Little is known about the prevalence and distribution of these E. coli in the animal production environment. An investigation of the prevalence of O157 and the six major non-O157 E. coli serogroups was conducted in 21 feedlots over the period July 2011 to October 2011. Individual fecal swabs were collected from cattle approximately 60 days after their arrival in the feedlot and were pooled for evaluation using a polymerase chain reaction assay to identify the presence of seven E. coli O-types (O157, O45, O103, O121, O145, O26, and O111) and four virulence genes (stx1, stx2, eaeA, and ehxA). Overall, 1145 fecal pools were evaluated, with 506 (44.2%) being positive for one or more of the E. coli O-serogroups. The pool prevalences for E. coli O157, O45, O26, O103, O121, O145, and O111 were 19.7%, 13.8%, 9.9%, 9.3%, 5.5%, 1.1%, and 0.5%, respectively. Nearly all pools were positive for ehxA (99.7%) or stx2 (98.6%). The pool level prevalence for stx1 and eae was 65.5% and 69.3%, respectively. Pools that were positive for one or more of the other E. coli O-serogroups were 1.37 times more likely to be positive for E. coli O157. Conversely, pools that were positive for E. coli O157 were 1.43 times more likely to be positive for at least one of the other E. coli O-serogroups evaluated. These data will be useful to understand the expected prevalence of potential Shiga toxin-producing E. coli in cattle feedlots.

Prevalence of Shiga toxin-producing Escherichia coli and associated virulence genes in feces of commercial feedlot cattle.

The objective of this study was to determine the prevalence of Shiga toxin-producing Escherichia coli (STEC) serogroups and associated virulence genes in feces of commercial feedlot cattle. During March to May 2011, fecal samples were collected from individual cattle (n=960) in 10 cohorts (cattle subpopulations within a feedlot) comprising 17,148 total steers that originated from 48 backgrounding operations in six U.S. states. Fecal samples were enriched in E. coli broth and subjected to two detection protocols: (1) an 11-gene multiplex polymerase chain reaction (PCR) that identifies seven O serogroups (O26, O45, O103, O111, O121, O145, and O157) and four virulence genes (stx1, stx2, eae, and ehxA) applied to extracted total DNA ("direct PCR"); and (2) cultural procedures that involve immunomagnetic separation (IMS) with O26, O103, and O111 beads, plating on a nondifferential MacConkey agar, followed by the multiplex PCR of pooled colonies ("culture-based method"). Generalized linear mixed models were used to adjust prevalence estimates for clustering. Based on direct PCR detection, O157 (49.9%) was the most prevalent O serogroup followed by O26 (20.3%), O103 (11.8%), O121 (10.7%), O45 (10.4%), O145 (2.8%), and O111 (0.8%). Cumulative adjusted prevalence estimates were 22.3, 24.6, and 0.01% for O26, O103, and O111 serogroups, respectively, based on culture-based methods. However, prevalence varied significantly by cohort (p-values<0.05) for O26, O121, and O157 based on direct PCR, and for O26, O103, and O111 serogroups based on culture-based methods. Results of this study indicate that all seven STEC serogroups were identified in feedlot cattle feces, with O157, O26, and O103 being the most prevalent serogroups. A substantial proportion of serogroup-positive samples did not harbor Shiga toxin genes; thus, additional elucidation of the potential human health risk is required. Further evaluation of diagnostic methods for non-O157 STEC is needed given their impact on prevalence estimation.

Digital Microfluidic Multiplex RT-qPCR for SARS-CoV-2 Detection and Variants Discrimination.

Continuous mutations have occurred in the genome of the SARS-CoV-2 virus since the onset of the COVID-19 pandemic. The increased transmissibility of the mutated viruses has not only imposed medical burdens but also prolonged the duration of the pandemic. A point-of-care (POC) platform that provides multitarget detection will help to track and reduce disease transmissions. Here we detected and discriminated three genotypes of SARS-CoV-2, including the wildtype and two variants of concern (VOCs), the Delta variant and Omicron variant, through reverse transcription quantitative polymerase chain reaction (RT-qPCR) on a digital microfluidics (DMF)-based cartridge. Upon evaluating with the RNA samples of Omicron variant, the DMF RT-qPCR presented a sensitivity of 10 copies/µL and an amplification efficiency of 96.1%, capable for clinical diagnosis. When spiking with SARS-CoV-2 RNA (wildtype, Delta variant, or Omicron variant) and 18S rDNA, the clinical analog samples demonstrated accurate detection and discrimination of different SARS-CoV-2 strains in 49 min.

High Nutritional Quality of Human-Induced Pluripotent Stem Cell-Generated Proteins through an Advanced Scalable Peptide Hydrogel 3D Suspension System.

Cell-cultured protein technology has become increasingly attractive due to its sustainability and climate benefits. The aim of this study is to determine the nutritional quality of the human-induced pluripotent stem cell (hiPSC)-cultured proteins in an advanced 3D peptide hydrogel system for the highly efficient production of cell-cultured proteins. Our previous study demonstrated a PGmatrix peptide hydrogel for the 3D embedded culture of long-term hiPSC maintenance and expansion (PGmatrix-hiPSC (PG-3D)), which showed significantly superior pluripotency when compared with traditional 2D cell culture on Matrigel and/or Vitronectin and other existing 3D scaffolding systems such as Polyethylene glycol (PEG)-based hydrogels. In this study, we designed a PGmatrix 3D suspension (PG-3DSUSP) system from the PG-3D embedded system that allows scaling up a hiPSC 3D culture volume by 20 times (e.g., from 0.5 mL to 10 mL). The results indicated that the PG-3DSUSP was a competitive system compared to the well-established PG-3D embedded method in terms of cell growth performance and cell pluripotency. hiPSCs cultured in PG-3DSUSP consistently presented a 15-20-fold increase in growth and a 95-99% increase in viability across multiple passages with spheroids with a size range of 30-50 µm. The expression of pluripotency-related genes, including NANOG, OCT4, hTERT, REX1, and UTF1, in PG-3DSUSP-cultured hiPSCs was similar to or higher than that observed in a PG-3D system, suggesting continuous pluripotent maintenance. The nutritional value of the hiPSC-generated proteins from the PG-3DSUSP system was further evaluated for amino acid composition and in vitro protein digestibility. The amino acid composition of the hiPSC-generated proteins demonstrated a significantly higher essential amino acid content (39.0%) than human skeletal muscle protein (31.8%). In vitro protein digestibility of hiPSC-generated proteins was significantly higher (78.0 ± 0.7%) than that of the commercial beef protein isolate (75.7 ± 0.6%). Taken together, this is the first study to report an advanced PG-3DSUSP culture system to produce highly efficient hiPSC-generated proteins that possess more essential amino acids and better digestibility. The hiPSC-generated proteins with superior nutrition quality may be of particular significance as novel alternative proteins in food engineering and industries for future food, beverage, and supplement applications.

Efficacy of high-level disinfection of endoscopes contaminated with Streptococcus equi subspecies equi with 2 different disinfectants.

BACKGROUND: Prevention of spread of Streptococcus equi subspecies equi (S. equi) after an outbreak is best accomplished by endoscopic lavage of the guttural pouch, with samples tested by culture and real time, quantitative polymerase chain reaction (qPCR). Disinfection of endoscopes must eliminate bacteria and DNA to avoid false diagnosis of carrier horses of S. equi. HYPOTHESIS/OBJECTIVES: Compare failure rates of disinfection of endoscopes contaminated with S. equi using 2 disinfectants (accelerated hydrogen peroxide [AHP] or ortho-phthalaldehyde [OPA]). The null hypothesis was that there would be no difference between the AHP and OPA products (based on culture and qPCR results) after disinfection. METHODS: Endoscopes contaminated with S. equi were disinfected using AHP, OPA or water (control). Samples were collected before and after disinfection and submitted for detection of S. equi by culture and qPCR. Using a multivariable logistic regression model-adjusted probability, with endoscope and day as controlled variables, the probability of an endoscope being qPCR-positive was determined. RESULTS: After disinfection, all endoscopes were culture-negative (0%). However, the raw unadjusted qPCR data were positive for 33% AHP, 73% OPA, and 71% control samples. The model-adjusted probability of being qPCR-positive after AHP disinfection was lower (0.31; 95% confidence interval [CI], -0.03-0.64) compared to OPA (0.81; 95% CI, 0.55-1.06), and control (0.72; 95% CI, 0.41-1.04). CONCLUSION AND CLINICAL IMPORTANCE: Disinfection using the AHP product resulted in significantly lower probability of endoscopes being qPCR-positive compared to the OPA product and control.

Validation of a real-time PCR panel for detection and quantification of nine pathogens commonly associated with canine infectious respiratory disease.

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.

Characterization of reference genes for quantitative real-time PCR analysis in various tissues of Anoectochilus roxburghii.

Accurate quantification of transcript profiling with quantitative real time polymerase chain reaction (qRT-PCR) relies on the reliable normalization of an appropriate reference gene. This study reported the identification and validation of nine reference genes, including ß-tubulin (ß-TUB), elongation factor 1 alpha (EF-1α), elongation factor 1 beta (EF-1ß), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin (UBQ), actin 1/2(ACT-1 and ACT-2), 18S rRNA, and 26S rRNA, from Anoectochilus roxburghii (Wall.) Lindl., a valuable herb remedy widely used for various diseases treatment in traditional Chinese medicine. Transcriptional levels of the candidate reference genes were examined using qRT-PCR analysis and revealed differential expression of the genes in the leaf, stem, root, flower, and peduncle tissues. The relative quantities data were subjected to geNorm software for ranking the expression stability of the reference genes and the results showed that EF-1ß and ACT-2 were the two best stable genes whereas GAPDH and 26S rRNA did not favor normalization of qRT-PCR in these tissues. The expression pattern of a squalene synthase encoding gene (SS) was also determined in parallel. The analyses were in great consistency when the qRT-PCR data was normalized to the expression of each or both of EF-1ß and ACT-2 as the internal control, further confirming the reliability of EF-1ß and ACT-2 as the best internal control. The present study provided the first important clues for accurate data normalization in transcript profiling in A. roxburghii, which will be essential to further functional genomics study in the valuable medicinal plant.