Transcriptome sequencing of the endangered land snail Karaftohelix adamsi from the Island Ulleung: De novo assembly, annotation, valuation of fitness genes and SSR markers.

BACKGROUND: The Bradybaenidae snail Karaftohelix adamsi is endemic to Korea, with the species tracked from Island Ulleung in North Gyeongsang Province of South Korea. K. adamsi has been classified under the Endangered Wildlife Class II species of Korea and poses a severe risk of extinction following habitat disturbances. With no available information at the DNA (genome) or mRNA (transcriptome) level for the species, conservation by utilizing informed molecular resources seems difficult. OBJECTIVE: In this study, we used the Illumina short-read sequencing and Trinity de novo assembly to draft the reference transcriptome of K. adamsi. RESULTS: After assembly, 13,753 unigenes were obtained of which 10,511 were annotated to public databases (a maximum of 10,165 unigenes found homologs in PANM DB). A total of 6,351, 3,535, 358, and 3,407 unigenes were ascribed to the functional categories under KOG, GO, KEGG, and IPS, respectively. The transcripts such as the HSP 70, aquaporin, TLR, and MAPK, among others, were screened as putative functional resources for adaptation. DNA transposons were found to be thickly populated in comparison to retrotransposons in the assembled unigenes. Further, 2,164 SSRs were screened with the promiscuous presence of dinucleotide repeats such as AC/GT and AG/CT. CONCLUSION: The transcriptome-guided discovery of molecular resources in K. adamsi will not only serve as a basis for functional genomics studies but also provide sustainable tools to be utilized for the protection of the species in the wild. Moreover, the development of polymorphic SSRs is valuable for the identification of species from newer habitats and cross-species genotyping.

Comparison of Cryopreservation Media for Mesenchymal Stem Cell Spheroids.

Multipotent mesenchymal stromal/stem cell (MSC) spheroids generated in three-dimensional culture are of considerable interest as a novel therapeutic tool for regenerative medicine. However, the lack of reliable methods for storing MSC spheroids represents a significant roadblock to their successful use in the clinic. An ideal storage medium for MSC spheroids should function as both a vehicle for delivery and a cryoprotectant during storage of spheroids for use at a later time. In this study, we compared the outcomes after subjecting MSC spheroids to a freeze/thaw cycle in three Good Manufacturing Practices-grade cryopreservation media, CryoStor10 (CS10), Stem-Cellbanker (SCB), and Recovery Cell Culture Freezing Media (RFM) or conventional freezing medium (CM) (CM, Dulbecco's modified Eagle's medium containing 20% fetal bovine serum and 10% dimethyl sulfoxide) as a control for 2 months. The endpoints tested were viability, morphology, and expression of stem cell markers and other relevant genes. The results of LIVE/DEAD™ assays and annexin V/propidium iodide staining suggested that viability was relatively higher after one freeze/thaw cycle in CS10 or SCB than after freeze/thaw in CM or RFM. Furthermore, the relative "stemness" and expression of MSC markers were similar with or without freeze/thaw in CS10. Scanning electron microscopy also indicated that the surface morphology of MSC spheroids was well preserved after cryopreservation in CS10. Thus, even though it was tested for a short-term period, we suggest that CS10, which has been approved for clinical use by the U.S. Food and Drug Association, is a promising cryopreservation medium that would facilitate the development of MSC spheroids for future clinical use.

Self-assembled adipose-derived mesenchymal stem cells as an extracellular matrix component- and growth factor-enriched filler.

The clinical application of mesenchymal stem cells (MSCs) is attracting attention due to their excellent safety, convenient acquisition, multipotency, and trophic activity. The clinical effectiveness of transplanted MSCs is well-known in regenerative and immunomodulatory medicine, but there is a demand for their improved viability and regenerative function after transplantation. In this study, we isolated MSCs from adipose tissue from three human donors and generated uniformly sized MSC spheroids (∼100 µm in diameter) called microblocks (MiBs) for dermal reconstitution. The viability and MSC marker expression of MSCs in MiBs were similar to those of monolayer MSCs. Compared with monolayer MSCs, MiBs produced more extracellular matrix (ECM) components, including type I collagen, fibronectin, and hyaluronic acid, and growth factors such as vascular endothelial growth factor and hepatocyte growth factor. Subcutaneously injected MiBs showed skin volume retaining capacity in mice. These results indicate that MiBs could be applied as regenerative medicine for skin conditions such as atrophic scar by having high ECM and bioactive factor expression.

Transcriptome analysis of Haemaphysalis flava female using Illumina HiSeq 4000 sequencing: de novo assembly, functional annotation and discovery of SSR markers.

BACKGROUND: Ticks are ectoparasites capable of directly damaging their hosts and transmitting vector-borne diseases. The ixodid tick Haemaphysalis flava has a broad distribution that extends from East to South Asia. This tick is a reservoir of severe fever with thrombocytopenia syndrome virus (SFTSV) that causes severe hemorrhagic disease, with cases reported from China, Japan and South Korea. Recently, the distribution of H. flava in South Korea was found to overlap with the occurrence of SFTSV. METHODS: This study was undertaken to discover the molecular resources of H. flava female ticks using the Illumina HiSeq 4000 system, the Trinity de novo sequence assembler and annotation against public databases. The locally curated Protostome database (PANM-DB) was used to screen the putative adaptation-related transcripts classified to gene families, such as angiotensin-converting enzyme, aquaporin, adenylate cyclase, AMP-activated protein kinase, glutamate receptors, heat shock proteins, molecular chaperones, insulin receptor, mitogen-activated protein kinase and solute carrier family proteins. Also, the repeats and simple sequence repeats (SSRs) were screened from the unigenes using RepeatMasker (v4.0.6) and MISA (v1.0) software tools, followed by the designing of SSRs flanking primers using BatchPrimer 3 (v1.0) software. RESULTS: The transcriptome produced a total of 69,822 unigenes, of which 46,175 annotated to the homologous proteins in the PANM-DB. The unigenes were also mapped to the EuKaryotic Orthologous Groups (KOG), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) specializations. Promiscuous presence of protein kinase, zinc finger (C2H2-type), reverse transcriptase, and RNA recognition motif domains was observed in the unigenes. A total of 3480 SSRs were screened, of which 1907 and 1274 were found as tri- and dinucleotide repeats, respectively. A list of primer sequences flanking the SSR motifs was detailed for validation of polymorphism in H. flava and the related tick species. CONCLUSIONS: The reference transcriptome information on H. flava female ticks will be useful for an enriched understanding of tick biology, its competency to act as a vector and the study of species diversity related to disease transmission.

A review of the endangered mollusks transcriptome under the threatened species initiative of Korea.

Transcriptome studies for conservation of endangered mollusks is a proactive approach towards managing threats and uncertainties facing these species in natural environments. The population of these species is declining due to habitat destruction, illicit wildlife trade, and global climate change. These activities risk the free movement of species across the wild landscape, loss of breeding grounds, and restrictions in displaying the physiological attributes so crucial for faunal welfare. Gastropods face the most negative ecological effects and have been enlisted under Korea's protective species consortium based on their population dynamics in the last few years. Moreover, with the genetic resources restricted for such species, conservation by informed planning is not possible. This review provides insights into the activities under the threatened species initiative of Korea with special reference to the transcriptome assemblies of endangered mollusks. The gastropods such as Ellobium chinense, Aegista chejuensis, Aegista quelpartensis, Incilaria fruhstorferi, Koreanohadra kurodana, Satsuma myomphala, and Clithon retropictus have been represented. Moreover, the transcriptome summary of bivalve Cristaria plicata and Caenogastropoda Charonia lampas sauliae is also discussed. Sequencing, de novo assembly, and annotation identified transcripts or homologs for the species and, based on an understanding of the biochemical and molecular pathways, were ascribed to predictive gene function. Mining for simple sequence repeats from the transcriptome have successfully assisted genetic polymorphism studies. A comparison of the transcriptome scheme of Korean endangered mollusks with the genomic resources of other endangered mollusks have been discussed with homologies and analogies for dictating future research.

A Transdiagnostic group therapy for sleep and anxiety among adults with substance use disorders: Protocol and pilot investigation.

Introduction: Treatment of substance use disorders (SUDs) is challenging with high rates of treatment dropout and relapse, particularly among individuals with comorbid psychiatric conditions. Anxiety and insomnia are prevalent among those with SUD and exacerbate poor treatment outcomes. Interventions that concurrently target anxiety and insomnia during the early stages of SUD treatment are lacking. To this end, we investigated the feasibility and preliminary effectiveness in a single-arm pilot trial of an empirically informed group transdiagnostic intervention, Transdiagnostic SUD Therapy, to concurrently reduce anxiety and improve sleep among adults receiving treatment for SUD. Specifically, we hypothesized that participants would evidence declines in anxiety and insomnia and improvements in sleep health, a holistic, multidimensional pattern of sleep-wakefulness that promotes wellbeing. A secondary aim was to describe the protocol for Transdiagnostic SUD Therapy and how it may be implemented into a real-world addiction treatment setting. Method: Participants were 163 adults (Mage = 43.23; 95.1% White; 39.93% female) participating in an intensive outpatient program for SUD who attended at least three of four Transdiagnostic SUD Therapy sessions. Participants had diverse SUDs (58.3% alcohol use disorder, 19.0% opioid use disorder) and nearly a third of the sample met criteria for two SUDs and comorbid mental health diagnoses (28.9% anxiety disorder, 24.6% major depressive disorder). Results: As anticipated, anxiety and insomnia reduced significantly across the 4-week intervention period from clinical to subclinical severity, and sleep health significantly improved (ps < 0.001). These statistically significant improvements following Transdiagnostic SUD Therapy demonstrated medium to large effects (ds > 0.5). Conclusion: Transdiagnostic SUD Therapy is designed to be flexibly administered in "real-world" clinical settings and, preliminarily, appears to be effective in improving emotional and behavioral factors that increase risk for return to substance use and poor SUD treatment outcomes. Additional work is needed to replicate these findings, determine the feasibility of widespread uptake of Transdiagnostic SUD Therapy, and examine whether the treatment effects translate to improvement in substance use outcomes.

Transcriptome analysis of the endangered dung beetle Copris tripartitus (Coleoptera: Scarabaeidae) and characterization of genes associated to immunity, growth, and reproduction.

BACKGROUND: Dung beetles recycle organic matter through the decomposition of feces and support ecological balance. However, these insects are threatened by the indiscriminate use of agrochemicals and habitat destruction. Copris tripartitus Waterhouse (Coleoptera: Scarabaeidae), a dung beetle, is listed as a class-II Korean endangered species. Although the genetic diversity of C. tripartitus populations has been investigated through analysis of mitochondrial genes, genomic resources for this species remain limited. In this study, we analyzed the transcriptome of C. tripartitus to elucidate functions related to growth, immunity and reproduction for the purpose of informed conservation planning. RESULTS: The transcriptome of C. tripartitus was generated using next-generation Illumina sequencing and assembled de novo using a Trinity-based platform. In total, 98.59% of the raw sequence reads were processed as clean reads. These reads were assembled into 151,177 contigs, 101,352 transcripts, and 25,106 unigenes. A total of 23,450 unigenes (93.40%) were annotated to at least one database. The largest proportion of unigenes (92.76%) were annotated to the locally curated PANM-DB. A maximum of 5,512 unigenes had homologous sequences in Tribolium castaneum. Gene Ontology (GO) analysis revealed a maximum of 5,174 unigenes in the Molecular function category. Further, in Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, a total of 462 enzymes were associated with established biological pathways. Based on sequence homology to known proteins in PANM-DB, representative immunity, growth, and reproduction-related genes were screened. Potential immunity-related genes were categorized into pattern recognition receptors (PRRs), the Toll-like receptor signaling pathway, the MyD88- dependent pathway, endogenous ligands, immune effectors, antimicrobial peptides, apoptosis, and adaptation-related transcripts. Among PRRs, we conducted detailed in silico characterization of TLR-2, CTL, and PGRP_SC2-like. Repetitive elements such as long terminal repeats, short interspersed nuclear elements, long interspersed nuclear elements and DNA elements were enriched in the unigene sequences. A total of 1,493 SSRs were identified among all unigenes of C. tripartitus. CONCLUSIONS: This study provides a comprehensive resource for analysis of the genomic topography of the beetle C. tripartitus. The data presented here clarify the fitness phenotypes of this species in the wild and provide insight to support informed conservation planning.

Pathogenesis of a novel porcine parainfluenza virus type 1 isolate in conventional and colostrum deprived/caesarean derived pigs.

Two experimental challenge studies were conducted to evaluate the pathogenesis of a porcine parainfluenza virus type 1 (PPIV-1) isolate. Four-week-old conventional (CON) pigs were challenged in Study 1 and six-week-old caesarean derived/colostrum deprived (CDCD) pigs were challenged in Study 2. Results indicate that PPIV-1 shedding and replication occur in the upper and lower respiratory tracts of CON and CDCD pigs as detected by RT-qPCR and immunohistochemistry. Mild macroscopic lung lesions were observed in CON pigs but not in CDCD pigs. Microscopic lung lesions were mild and consisted of peribronchiolar lymphocytic cuffing and epithelial proliferation in CON and CDCD pigs. Serum neutralizing antibodies were detected in the CON and CDCD pigs by 14 and 7 days post inoculation, respectively. This study provides evidence that in spite of PPIV-1 infection and replication in challenged swine, significant clinical respiratory disease was not observed.

De novo transcriptome sequencing of triton shell Charonia lampas sauliae: Identification of genes related to neurotoxins and discovery of genetic markers.

Charonia lampas sauliae (triton snails, triton shells or tritons; Mollusca, Caenogastropoda, Littorinimorpha, Ranellidae) is a marine species with a wide distribution. In Korea, this species is listed as vulnerable and is regionally protected as an endangered species. Here, we report the first comprehensive transcriptome dataset of C. lampas sauliae obtained using the Illumina HiSeq 2500 platform. In total, 97.68% of raw read sequences were processed as clean reads. Of the 577,478 contigs obtained, 146,026 sequences were predicted to contain coding regions. About 89.34% of all annotated unigene sequences showed homologous matches to protein sequences in PANM DB (Protostome database). Further, about one-third of the unigene sequences were annotated using the UniGene, Swiss-Prot, Clusters of Orthologous Groups (COG) and Gene Ontology (GO) databases. In total, 190 enzymes were predicted under key metabolic pathways under stood through Kyoto Encyclopedia of Genes and Genomes (KEGG) database annotation. Repetitive elements such as long terminal repeats (LTRs), short interspersed nuclear elements (SINEs), long interspersed nuclear elements (LINEs), and DNA elements were enriched in the unigene sequences. Among the identified transcripts were the channel proteins, some of which were blocked by tetrodotoxin, which is thought to be synthesized by symbiotic bacteria inhabiting the shells. In addition, conotoxin superfamily peptides, such as B-conotoxin, conotoxin superfamily T and alpha-conotoxin, were identified, which may have relevance to biomedical and evolutionary research. A transcriptome-wide search for polymorphic loci identified 21,568 simple sequence repeats (SSRs) in the unigene sequences. Most SSRs were dinucleotides, among which AC/GT was the dominant SSR type. The molecular and genetic resources revealed in this study could be utilized for investigations on the fitness of the species in the marine environment and sustainability in a changing habitat.

Emergence of new phylogenetic lineage of Influenza D virus with broad antigenicity in California, United States.

Influenza D virus (IDV), with bovines as a primary host, circulates widely in cattle populations across North America and Eurasia. Here we report the identification of a novel IDV group with broad antigenicity in U.S. bovine herds, which is genetically different from previously known lineages of IDV.

Comparison of ZMAC and MARC-145 Cell Lines for Improving Porcine Reproductive and Respiratory Syndrome Virus Isolation from Clinical Samples.

The MARC-145 cell line is commonly used to isolate porcine reproductive and respiratory syndrome virus (PRRSV) for diagnostics, research, and vaccine production, but it yields frustratingly low success rates of virus isolation (VI). The ZMAC cell line, derived from porcine alveolar macrophages, has become available, but its utilization for PRRSV VI from clinical samples has not been evaluated. This study compared PRRSV VI results in ZMAC and MARC-145 cells from 375 clinical samples (including 104 lung, 140 serum, 90 oral fluid, and 41 processing fluid samples). The PRRSV VI success rate was very low in oral fluids and processing fluids regardless of whether ZMAC cells or MARC-145 cells were used. Success rates of PRRSV VI from lung and serum samples were significantly higher in ZMAC than in MARC-145 cells. Lung and serum samples with threshold cycle (CT ) values of <30 had better VI success. PRRSV-2 in genetic lineages 1 and 8 was isolated more successfully in ZMAC cells than in MARC-145 cells, whereas PRRSV-2 in genetic lineage 5 was isolated in the two cell lines with similar success rates. For samples with positive VI in both ZMAC and MARC-145 cells, 14 of 23 PRRSV-2 isolates had similar titers in the two cell lines. A total of 51 of 95 (53.7%) ZMAC-obtained PRRSV-2 or PRRSV-1 isolates grew in MARC-145 cells, and all 46 (100%) MARC-145-obtained isolates grew in ZMAC cells. In summary, ZMAC cells allow better isolation of a wide range of PRRSV field strains; however, not all of the ZMAC-obtained PRRSV isolates grow in MARC-145 cells. This report provides important guidelines to improve isolation of PRRSV from clinical samples for further characterization and/or for producing autogenous vaccines.

Transcriptome analysis of air-breathing land slug, Incilaria fruhstorferi reveals functional insights into growth, immunity, and reproduction.

BACKGROUND: Incilaria (= Meghimatium) fruhstorferi is an air-breathing land slug found in restricted habitats of Japan, Taiwan and selected provinces of South Korea (Jeju, Chuncheon, Busan, and Deokjeokdo). The species is on a decline due to depletion of forest cover, predation by natural enemies, and collection. To facilitate the conservation of the species, it is important to decide on a number of traits related to growth, immunity and reproduction addressing fitness advantage of the species. RESULTS: The visceral mass transcriptome of I. fruhstorferi was enabled using the Illumina HiSeq 4000 sequencing platform. According to BUSCO (Benchmarking Universal Single-Copy Orthologs) method, the transcriptome was considered complete with 91.8% of ortholog genes present (Single: 70.7%; Duplicated: 21.1%). A total of 96.79% of the raw read sequences were processed as clean reads. TransDecoder identified 197,271 contigs that contained candidate-coding regions. Of a total of 50,230 unigenes, 34,470 (68.62% of the total unigenes) annotated to homologous proteins in the Protostome database (PANM-DB). The GO term and KEGG pathway analysis indicated genes involved in metabolism, phosphatidylinositol signalling system, aminobenzoate degradation, and T-cell receptor signalling pathway. Many genes associated with molluscan innate immunity were categorized under pathogen recognition receptor, TLR signalling pathway, MyD88 dependent pathway, endogenous ligands, immune effectors, antimicrobial peptides, apoptosis, and adaptation-related. The reproduction-associated unigenes showed homology to protein fem-1, spermatogenesis-associated protein, sperm associated antigen, and testis expressed sequences, among others. In addition, we identified key growth-related genes categorized under somatotrophic axis, muscle growth, chitinases and collagens. A total of 4822 Simple Sequence Repeats (SSRs) were also identified from the unigene sequences of I. fruhstorferi. CONCLUSIONS: This is the first available genomic information for non-model land slug, I. fruhstorferi focusing on genes related to growth, immunity, and reproduction, with additional focus on microsatellites and repeating elements. The transcriptome provides access to greater number of traits of unknown relevance in the species that could be exploited for in-depth analyses of evolutionary plasticity and making informed choices during conservation planning. This would be appropriate for understanding the dynamics of the species on a priority basis considering the ecological, health, and social benefits.

Detection, isolation, and in vitro characterization of porcine parainfluenza virus type 1 isolated from respiratory diagnostic specimens in swine.

Porcine parainfluenza virus type 1 (PPIV-1) is a member of the genus Respirovirus in the family Paramyxoviridae. The PPIV-1 was initially detected in 2013 from slaughter pigs in Hong Kong, China although its role in respiratory disease has remained unknown without virus isolates for experimental inoculation in swine. The objective of this study was to determine the relative frequency of PPIV-1 detection in diagnostic samples collected from swine in the United States, describe the cell culture isolation of PPIV-1, and characterize PPIV-1 cell culture isolates in vitro. Among 842 porcine specimens submitted to the Iowa State University Veterinary Diagnostic Laboratory during 2016-2017, 43.3% were PPIV-1 positive by a real-time, reverse transcriptase PCR suggesting PPIV-1 may be common in swine. Two strains of PPIV-1 were successfully isolated in an LLC-MK2 cell line from a PPIV-1 RT-qPCR positive nasal swab (USA/MN25890NS/2016) and lung (USA/IA84915LG/2017). The PPIV-1 cytopathic effect was demonstrated in tissue culture and enveloped viral particles were observed by electron microscopy. The whole genome, F, and HN gene sequences of both isolates share 98.2%, 98.5%, and 98.2% nucleotide homology, respectively, and phylogenetic analysis indicated they are closely related to other PPIV-1 strains detected in swine from the United States. Whole virus PPIV-1-specific monoclonal antibodies were generated for PPIV-1 detection in infected LLC-MK2 cells by indirect immunofluorescence and immunocytochemistry assays. The virus isolates and monoclonal antibodies obtained in the present study can be used to investigate the pathogenesis of PPIV-1 and develop new diagnostic tests.

Transcriptome analysis of the threatened snail Ellobium chinense reveals candidate genes for adaptation and identifies SSRs for conservation genetics.

Ellobium chinense (Pfeiffer, 1854) is a brackish pulmonate species that inhabits the bases of mangrove trees and is most commonly found in salt grass meadows. Threats to mangrove ecosystems due to habitat degradation and overexploitation have threatened the species with extinction. In South Korea, E. chinense has been assessed as vulnerable, but there are limited data on its population structure and distribution. The nucleotide and protein sequences for this species are not available in databases, which limits the understanding of adaptation-related traits. We sequenced an E. chinense cDNA library using the Illumina platform, and the subsequent bioinformatics analysis yielded 227,032 unigenes. Of these unigenes, 69,088 were annotated to matched protein and nucleotide sequences in databases, for an annotation rate of 30.42%. Among the predominant gene ontology terms, cellular and metabolic processes (under the biological process category), membrane and cell (under the cellular component category), and binding and catalytic activity (under the molecular function category) were noteworthy. In addition, 4850 unigenes were distributed to 15 Kyoto Encyclopaedia of Genes and Genomes based enrichment categories. Among the candidate genes related to adaptation, angiotensin I converting enzyme, adenylate cyclase activating polypeptide, and AMP-activated protein kinase were the most prominent. A total of 15,952 simple sequence repeats (SSRs) were identified in sequences of > 1 kb in length. The di- and trinucleotide repeat motifs were the most common. Among the repeat motif types, AG/CT, AC/GT, and AAC/GTT dominated. Our study provides the first comprehensive genomics dataset for E. chinense, which favors conservation programs for the restoration of the species and provides sufficient evidence for genetic variability among the wild populations.

Long-term safety of bioresorbable scaffolds: insights from a network meta-analysis including 91 trials.

AIMS: The aim of this study was to investigate the long-term safety and efficacy of biodegradable scaffolds and metallic stents. METHODS AND RESULTS: We analysed a total of 91 randomised controlled trials with a mean follow-up of 3.7 years in 105,842 patients which compared two or more coronary metallic stents or biodegradable scaffolds and reported the long-term clinical outcomes (≥2 years). Network meta-analysis showed that patients treated with the Absorb bioresorbable vascular scaffold (BVS) had a significantly higher risk of definite or probable scaffold thrombosis (ScT) compared to those treated with metallic DES. The risk of very late ScT was highest with the Absorb BVS among comparators. Pairwise conventional meta-analysis demonstrated that the elevated risk of ScT with Absorb BVS compared to cobalt-chromium everolimus-eluting stents was consistent across the time points of ≤30 days (early), 31 days - 1 year (late) and >1 year (very late) ScT. In addition, target lesion failure rates were significantly higher in the Absorb BVS cohort, driven by both increased risk of target vessel myocardial infarction and ischaemia-driven target lesion revascularisation. CONCLUSIONS: Absorb BVS implantation was associated with increased risk of long-term and very late ScT compared to current-generation metallic DES. The risk of ScT occurred with a rising trend beyond one year.

Complete Genome Sequence of Porcine respirovirus 1 Strain USA/MN25890NS/2016, Isolated in the United States.

A porcine parainfluenza virus type 1 (species Porcine respirovirus 1) cell culture isolate, USA/MN25890NS/2016, was obtained from porcine nasal swabs, and its complete genome sequence (GenBank accession number MF681710) was determined to help further characterize this virus.

Concomitant origin of the anterior or posterior spinal artery with the feeder of a spinal dural arteriovenous fistula (SDAVF).

BACKGROUND/OBJECTIVE: The concomitant origin of the anterior spinal artery (ASA) or the posterior spinal artery (PSA) from the feeder of a spinal dural arteriovenous fistula (SDAVF) is rare and the exact incidence is not known. We present our experience with the management of SDAVFs in such cases. METHODS: In 63 patients with SDAVF between 1993 and 2015, the feeder origin of the SDAVF was evaluated to determine whether it was concomitant with the origin of the ASA or PSA. Embolization was attempted when the patient did not want open surgery and an endovascular approach was regarded as safe and possible. The outcome of the procedure was evaluated as complete, partial, or no obliteration. The clinical outcome was evaluated by Aminoff-Logue (ALS) gait and micturition scale scores. RESULTS: Nine patients (14%) had a concomitant origin of the ASA or PSA with the feeder. There were two cervical, five thoracic, and two lumbar level SDAVFs. A concomitant origin of the feeder was identified with the ASA (n=7) and PSA (n=2). Embolization was performed in four patients and open surgery was performed in five. Embolization resulted in complete obliteration in three patients and partial obliteration in one. Using the ALS gait and micturition scale, the final outcome improved in six while three cases remained in an unchanged condition over 2-148 months. CONCLUSIONS: The concomitant origin of the ASA or PSA with the feeder occurs occasionally. Complete obliteration of the fistula can be achieved either by embolization or open surgery. Embolization can be carefully performed in selected patients who are in a poor condition and do not want to undergo open surgery.

Protection Against Necrotic Enteritis in Broiler Chickens by Regulated Delayed Lysis Salmonella Vaccines.

Necrotic enteritis (NE), caused by Gram-positive Clostridium perfringens type A strains, has gained more attention in the broiler industry due to governmental restrictions affecting the use of growth-promoting antibiotics in feed. To date, there is only one commercial NE vaccine available, based on the C. perfringens alpha toxin. However, recent work has suggested that the NetB toxin, not alpha toxin, is the most critical virulence factor for causing NE. These findings notwithstanding, it is clear from prior research that immune responses against both toxins can provide some protection against NE. In this study, we delivered a carboxyl-terminal fragment of alpha toxin and a GST-NetB fusion protein using a novel attenuated Salmonella vaccine strain designed to lyse after 6-10 rounds of replication in the chicken host. We immunized birds with vaccine strains producing each protein individually, a mixture of the two strains, or with a single vaccine strain that produced both proteins. Immunization with strains producing either of the single proteins was not protective, but immunization with a mixture of the two or with a single strain producing both proteins resulted in protective immunity. The vaccine strain synthesizing both PlcC and GST-NetB was able to elicit strong production of intestinal IgA, IgY, and IgM antibodies and significantly protect broilers against C. perfringens challenge against both mild and severe challenges. Although not part of our experimental plan, the broiler chicks we obtained for these studies were apparently contaminated during transit from the hatchery with group D Salmonella. Despite this drawback, the vaccines worked well, indicating applicability to real-world conditions.

Inflammatory effects of Edwardsiella ictaluri lipopolysaccharide modifications in catfish gut.

Bacterial lipopolysaccharides (LPS) are structural components of the outer membranes of Gram-negative bacteria and also are potent inducers of inflammation in mammals. Higher vertebrates are extremely sensitive to LPS, but lower vertebrates, like fish, are resistant to their systemic toxic effects. However, the effects of LPS on the fish intestinal mucosa remain unknown. Edwardsiella ictaluri is a primitive member of the Enterobacteriaceae family that causes enteric septicemia in channel catfish (Ictalurus punctatus). E. ictaluri infects and colonizes deep lymphoid tissues upon oral or immersion infection. Both gut and olfactory organs are the primary sites of invasion. At the systemic level, E. ictaluri pathogenesis is relatively well characterized, but our knowledge about E. ictaluri intestinal interaction is limited. Recently, we observed that E. ictaluri oligo-polysaccharide (O-PS) LPS mutants have differential effects on the intestinal epithelia of orally inoculated catfish. Here we evaluate the effects of E. ictaluri O-PS LPS mutants by using a novel catfish intestinal loop model and compare it to the rabbit ileal loop model inoculated with Salmonella enterica serovar Typhimurium LPS. We found evident differences in rabbit ileal loop and catfish ileal loop responses to E. ictaluri and S. Typhimurium LPS. We determined that catfish respond to E. ictaluri LPS but not to S. Typhimurium LPS. We also determined that E. ictaluri inhibits cytokine production and induces disruption of the intestinal fish epithelia in an O-PS-dependent fashion. The E. ictaluri wild type and ΔwibT LPS mutant caused intestinal tissue damage and inhibited proinflammatory cytokine synthesis, in contrast to E. ictaluri Δgne and Δugd LPS mutants. We concluded that the E. ictaluri O-PS subunits play a major role during pathogenesis, since they influence the recognition of the LPS by the intestinal mucosal immune system of the catfish. The LPS structure of E. ictaluri mutants is needed to understand the mechanism of interaction.

Multiplex Real-time Polymerase Chain Reaction Assays for Simultaneous Detection of Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus.

OBJECTIVES: A multiplex real-time polymerase chain reaction (RT-PCR) method was developed for the identification of three Vibrio species: Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus. METHODS: Specific primers and probes targeting the hlyA, tlh, and vvhA genes were selected and used for multiplex real-time PCR to confirm the identification of V. cholerae, V. parahaemolyticus, and V. vulnificus, respectively. This method was applied to screen Vibrio species from environmental samples and combining it with a culture-based method, its effectiveness was evaluated in comparison with culture-based methods alone. RESULTS: Specific PCR fragments were obtained from isolates belonging to the target species, indicating a high specificity of this multiplex real-time PCR. No cross-reactivity with the assay was observed between the tested bacteria. The sensitivity of the multiplex real-time PCR was found to have a lower limit of 10(4) colony-forming units/reaction for all three Vibrio species. The combination strategy raised the isolation ratio of all three Vibrio species 1.26- to 2.75-fold. CONCLUSION: This assay provides a rapid, sensitive, and specific technique to detect these three Vibrio species in the environment.