Multi-omics analysis of miRNA-mediated intestinal microflora changes in crucian carp Carassius auratus infected with Rahnella aquatilis.

Infection by an emerging bacterial pathogen Rahnella aquatilis caused enteritis and septicemia in fish. However, the molecular pathogenesis of enteritis induced by R. aquatilis infection and its interacting mechanism of the intestinal microflora associated with microRNA (miRNA) immune regulation in crucian carp Carassius auratus are still unclear. In this study, C. auratus intraperitoneally injected with R. aquatilis KCL-5 was used as an experimental animal model, and the intestinal pathological changes, microflora, and differentially expressed miRNAs (DEMs) were investigated by multi-omics analysis. The significant changes in histopathological features, apoptotic cells, and enzyme activities (e.g., lysozyme (LYS), alkaline phosphatase (AKP), alanine aminotransferase (ALT), aspartate transaminase (AST), and glutathione peroxidase (GSH-Px)) in the intestine were examined after infection. Diversity and composition analysis of the intestinal microflora clearly demonstrated four dominant bacteria: Proteobacteria, Fusobacteria, Bacteroidetes, and Firmicutes. A total of 87 DEMs were significantly screened, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the potential target genes were mainly involved in the regulation of lipid, glutathione, cytosine, and purine metabolism, which participated in the local immune response through the intestinal immune network for IgA production, lysosome, and Toll-like receptor (TLR) pathways. Moreover, the expression levels of 11 target genes (e.g., TLR3, MyD88, NF-κB, TGF-ß, TNF-α, MHC II, IL-22, LysC, F2, F5, and C3) related to inflammation and immunity were verified by qRT-PCR detection. The correlation analysis indicated that the abundance of intestinal Firmicutes and Proteobacteria was significantly associated with the high local expression of miR-203/NF-κB, miR-129/TNF-α, and miR-205/TGF-ß. These findings will help to elucidate the molecular regulation mechanism of the intestinal microflora, inflammation, and immune response-mediated miRNA-target gene axis in cyprinid fish.

Single-cell transcriptome, phagocytic activity and immunohistochemical analysis of crucian carp (Carassius auratus) in response to Rahnella aquatilis infection.

In teleost fish, kidney is an important immune and hematopoietic organ with multiple physiological functions. However, the immune cells and cellular markers of kidney require further elucidation in crucian carp (C. auratus). Here we report on the single-cell transcriptional landscape in posterior kidney, immunohistochemical and phagocytic features of C. auratus with R. aquatilis infection. The results showed that a total of 18 cell populations were identified for the main immune cells such as monocytes/macrophages (Mo/Mφ), dendritic cells (DCs), B cells, T cells, granulocytes and hematopoietic progenitor cells (HPCs). Pseudo-time trajectory analysis was reconstructed for the immune cells using Monocle2 to obtain additional insights into their developmental lineage relationships. In the detected tissues (liver, spleen, kidney, intestine, skin, and gills) of infected fish exhibited positive immunohistochemical staining with prepared for antibody to R. aquatilis. Apoptotic cells were fluorescently demonstrated by TUNEL assay, and bacterial phagocytic activity were observed for neutrophils and Mo/Mφ cells, respectively. Moreover, a similar up-ward/down-ward expression trend of the selected immune and inflammatory genes was found in the kidney against R. aquatilis infection, which were significantly involved in TLR/NLR, ECM adhesion, phago-lysosome, apoptosis, complement and coagulation pathways. To our knowledge, this is the first report on the detailed characterization of immune cells and host-R. aquatilis interaction, which will contribute to understanding on the biology of renal immune cells and repertoire of potential markers in cyprinid fish species.

Focal adhesion signaling pathway involved in skin immune response of tongue sole Cynoglossus semilaevis to Vibrio vulnificus infection.

Focal adhesion (FA) plays a key role in cell adhesion, migration and antibacterial immune, but it remained unclear in fish. In this study, half-smooth tongue sole Cynoglossus semilaevis were infected with Vibrio vulnificus, and then immune-related protein in the skin, especially for FA signaling pathway were screened and identified by iTRAQ analysis. Results showed that the differentially expressed proteins (DEPs) in skin immune response (eg., ITGA6, FN, COCH, AMBP, COL6A1, COL6A3, COL6A6, LAMB1, LAMC1, FLMNA) were firstly found in FA signaling pathway. Furthermore, the validation analysis of FA-related genes were basically consistent with the iTRAQ data at 36 hpi (r = 0.678, p < 0.01), and their spatio-temporal expressions were confirmed by qPCR analysis. The molecular characterization of vinculin of C. semilaevis was described. This study will provide a new perspective for understanding the molecular mechanism of FA signaling pathway in the skin immune response in marine fish.

Multiomics analysis revealed miRNAs as potential regulators of the immune response in Carassius auratus gills to Aeromonas hydrophila infection.

The gill of fish is an important immune organ for pathogen defense, but its microRNA (miRNA) expression and regulatory mechanism remain unclear. In this study, we report on the histopathological and immunohistochemical features of the gills of the crucian carp Carassius auratus challenged with Aeromonas hydrophila. Small RNA libraries of the gills were constructed and sequenced on the Illumina HiSeq 2000 platform. A total of 1,165 differentially expressed miRNAs (DEMs) were identified in gills, of which 539 known and 7 unknown DEMs were significantly screened (p < 0.05). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the potential target genes/proteins were primarily involved in 33 immune-related pathways, in which the inflammatory responses were focused on the Toll-like receptor (TLR), mitogen-activated protein kinase (MAPK), and nuclear factor kappa B (NF-κB) signaling pathways. Moreover, the expression levels of 14 key miRNAs (e.g., miR-10, miR-17, miR-26a, miR-144, miR-145, and miR-146a) and their target genes (e.g., TNFα, TLR4, NF-κB, TAB1, PI3K, and IRAK1) were verified. In addition, the protein levels based on isobaric tags for relative and absolute quantification (iTRAQ) were significantly associated with the results of the quantitative real-time PCR (qRT-PCR) analysis (p < 0.01). miR-17/pre-miR-17 were identified in the regulation expression of the NF-κB target gene, and the phylogenetic tree analysis showed that the pre-miR-17 of C. auratus with the closest similarity to the zebrafish Danio rerio is highly conserved in teleosts. This is the first report of the multi-omics analysis of the miRNAs and proteins in the gills of C. auratus infected with A. hydrophila, thus enriching knowledge on the regulation mechanism of the local immune response in Cyprinidae fish.

Comprehensive transcriptomics and proteomics analysis of Carassius auratus gills in response to Aeromonas hydrophila.

As one of the mucosal barriers, fish gills represent the first line of defense against pathogen infection. However, the exact mechanism of gill mucosal immune response to bacterial infection still needs further investigation in fish. Here, to investigate pathological changes and molecular mechanisms of the mucosal immune response in the gills of crucian carp (Carassius auratus) challenged by Aeromonas hydrophila, the transcriptomics and proteomics were performed by using multi-omics analyses of RNA-seq coupled with iTRAQ techniques. The results demonstrated gill immune response were mostly related to the activation of complement and coagulation cascades, antigen processing and presentation, phagosome, NOD-like receptor (NLR) and nuclear factor κB (NFκB) signaling pathway. Selected 21 immune-related DEGs (ie., Clam, nfyal, snrpf, acin1b, psme, sf3b5, rbm8a, rbm25, prpf18, g3bp2, snrpd3l, tecrem-2, cfl-A, C7, lysC, ddx5, hsp90, α-2M, C9, C3 and slc4a1a) were verified for their immune roles in the A. hydrophila infection via using qRT-PCR assay. Meanwhile, some complement (C3, C7, C9, CFD, DF and FH) and antigen presenting (HSP90, MHC Ⅱ, CALR, CANX and PSME) proteins were significantly participated in the process of defense against infections in gill tissues, and protein-protein interaction (PPI) network displayed the immune signaling pathways and interactions among these DEPs. The correlation analysis indicated that the iTRAQ and qRT-PCR results was significantly correlated (Pearson's correlation coefficient = 0.70, p < 0.01). To our knowledge, the transcriptomics and proteomics of gills firstly identified by multi-omics analyses contribute to understanding on the molecular mechanisms of local mucosal immunity in cyprinid species.

Development of a TaqMan real-time quantitative PCR assay to detect Metschnikowia bicuspidata in Chinese mitten crab Eriocheir sinensis.

Milky disease of Chinese mitten crab Eriocheir sinensis caused by Metschnikowia bicuspidata is a novel disease with high mortality. No effective treatment is currently available, but a rapid, accurate detection method is required for the prevention and control of the disease. In this study, the genome-sequencing results of M. bicuspidata and similar species were used for comparative genomic analysis for genes specific to M. bicuspidata. A quantitative PCR (qPCR) detection method for M. bicuspidata was then established using the specific primers and probes designed according to the sequence of a hypothetical protein gene specific to M. bicuspidata. The assay was found to have a high degree of repeatability and reproducibility, with a linear dynamic range (R2 = 0.998) extending over 9 log10 dilutions and a high efficiency (100.7%). Furthermore, the method showed high sensitivity, being able to detect at least 11.3 copies µl-1 of recombinant plasmid, and strong specificity, without any cross-reaction with any of the 9 species of yeast that are closely related to M. bicuspidata or any of 16 species of pathogenic bacteria commonly observed in aquatic animals. The established method was used to examine 138 apparently healthy crabs collected from 22 farms, with 21 samples (15.2%) found to be M. bicuspidata-positive. Thus, the developed qPCR assay is a specific, sensitive, stable, and rapid diagnostic method for the detection and quantification of M. bicuspidata DNA from E. sinensis tissues.

Skin immune response to Aeromonas hydrophila infection in crucian carp Carassius auratus revealed by multi-omics analysis.

Fish skin is an essential protective barrier and functions as the first line of immune defense against pathogens. However, the molecular mechanism at the proteome-level remains unclear in the skin of fish. In this study, the comparative proteomics of skin immune responses of crucian carp Carassius auratus infected with Aeromonas hydrophila was investigated by isobaric tags for relative and absolute quantification (iTRAQ), two-dimensional gel electrophoresis combined with mass spectrometry (2-DE/MS) as well as high-throughput transcriptome (RNA-seq) techniques. A total of 241 and 178 differentially expressed proteins (DEPs) at 6 and 12 h post-infection (hpi) were respectively identified by iTRAQ, and key-DEPs were furtherly verified with 2-DE/MS analysis. GO and KEGG analysis showed that these DEPs were mostly related to metabolism, regulation of the cytoskeleton, stress and immune responses. Co-association results of proteome and transcriptome revealed the lysozyme (LYZ), complement C3, DnaJ (Hsp40) homolog subfamily C member 8 (DNAJC8) and allograft inflammatory factor 1-like (AIF1L) play important roles in skin immune responses of crucian carp. The significantly up-regulated expression of detected immune-related genes (c3, mapk3, f5, nlr, hsp90, itgb2, fnl, flnca, p47, mhc and pros1) were validated by qRT-PCR analysis. To our knowledge, this is first report on multi-omics analysis of the differential proteomics for the skin immune response of C. auratus against A.hydrophila infection, which contribute to the understanding the mechanisms of skin mucosal immunity in cyprinid fish.

First record of isolation and characterization of Vibrio sinaloensis from diseased orange-spotted grouper Epinephelus coioides.

A dominant bacterium, ZYL-12, isolated from the liver of a diseased orange-spotted grouper Epinephelus coioides, was identified as Vibrio sinaloensis, based on phenotypic and molecular analysis. The median lethal dosage of ZYL-12 was calculated as 1.6 × 105 CFU g-1 fish weight. The infection experiment indicated that ZYL-12 caused noticeable histological lesions to the liver, kidney and spleen of the fish. Growth characteristics showed that ZYL-12 possessed strong environmental adaptability. This note is the first report about the pathogenicity of V. sinaloensis isolated from diseased fish.

Phenotypic and genomic characterization of a Vibrio parahaemolyticus strain causing disease in Penaeus vannamei provides insights into its niche adaptation and pathogenic mechanism.

The virulence of Vibrio parahaemolyticus is variable depending on its virulence determinants. A V. parahaemolyticus strain, in which the virulence is governed by the pirA and pirB genes, can cause acute hepatopancreatic necrosis disease (AHPND) in shrimps. Some V. parahaemolyticus that are non-AHPND strains also cause shrimp diseases and result in huge economic losses, while their pathogenicity and pathogenesis remain unclear. In this study, a non-AHPND V. parahaemolyticus, TJA114, was isolated from diseased Penaeus vannamei associated with a high mortality. To understand its virulence and adaptation to the external environment, whole-genome sequencing of this isolate was conducted, and its phenotypic profiles including pathogenicity, growth characteristics and nutritional requirements were investigated. Shrimps following artificial infection with this isolate presented similar clinical symptoms to the naturally diseased ones and generated obvious pathological lesions. The growth characteristics indicated that the isolate TJA114 could grow well under different salinity (10-55 p.p.t.), temperature (23-37 °C) and pH (6-10) conditions. Phenotype MicroArray results showed that this isolate could utilize a variety of carbon sources, amino acids and a range of substrates to help itself adapt to the high hyperosmotic and alkaline environments. Antimicrobial-susceptibility test showed that it was a multidrug-resistant bacterium. The whole-genomic analysis showed that this V. parahaemolyticus possessed many important functional genes associated with multidrug resistance, stress response, adhesions, haemolysis, putative secreted proteases, dedicated protein secretion systems and a variety of nutritional metabolic mechanisms. These annotated functional genes were confirmed by the phenotypic profiles. The results in this study indicated that this V. parahaemolyticus isolate possesses a high pathogenicity and strong environmental adaptability.

Differentially expressed proteins in the intestine of Cynoglossus semilaevis Günther following a Shewanella algae challenge.

Fish intestine is an important constituent of the mucosal immune system. The gut and gut-associated lymphoid tissue construct a local immune environment. A Shewanella algae strain was previously reported to be a pathogen causing ascitic disease accompanied with intestinal inflammation in Cynoglossus semilaevis. This study aimed to investigate the intestine immune response in C. semilaevis to S. algae infection at the protein level. Two-dimensional electrophoresis coupled with mass spectrometry proteomics was utilized to compare protein expression in the intestines from normal and S. algae-infected C. semilaevis. A total of 70 differentially expressed proteins (DEPs), consisting of 16 upregulated and 54 downregulated proteins, were identified in the intestine tissue of C. Semilaevis. These protein expression changes were further validated using western blot analysis and quantitative real-time PCR. Gene ontology enrichment analysis showed that these 70 DEPs could be assigned across three categories: "cellular components", "molecular function", and "biological process". Forty-one DEPs (six up-regulated and 35 down-regulated proteins) related to metabolic processes were identified. In addition, 20 DEPs (eight up-regulated and 12 down-regulated proteins) related to stress and immune responses were identified. A protein-protein interaction network generated by the STRING (Search Tool for the Retrieval of Interacting Genes/protein) revealed that 30 DEPs interacted with one another to form an integrated network. Among them, 29 DEPs were related to stress, immune, and metabolism processes. In the network, some of the immune related proteins (C9, FGB, KNG1, apolipoprotein A-IV-like, and PDIA3) were up-regulated and most DEPs involved in metabolism processes were down-regulated. These results indicate that the immune defense response of the intestine was activated and the intestinal function associated with metabolism processes was disturbed. This study provides valuable information for further research into the functions of these DEPs in fish.

A candidate probiotic strain of Enterococcus faecium from the intestine of the crucian carp Carassius auratus.

In the present study, a Gram-positive bacterium was isolated from the intestine of healthy crucian carp Carassius auratus and named strain R8. It was initially identified as Enterococcus faecium according to its morphological, physiological and biochemical characteristics. Further identification by using 16S rRNA gene sequence analysis confirmed the R8 strain (Genbank accession no. MF928076) as E. faecium. Challenge and hemolysis experiments showed that the E. faecium R8 strain had no toxicity to the crucian carp. Bacteriostatic experiment showed that this isolate obviously inhibited the growth of Aeromonas veronii and Staphylococcus haemolyticus. The proliferation of E. faecium R8 strain occurred after exposure to various growth conditions such as at pH values from 2.0 to 4.0 for 8 h, bile concentrations from 0.2 to 1.2% and high temperature of 80 °C. This bacterial strain grew best under the condition of 37 °C, pH 7.0 and salinity 30 ppt, and its growth curve exhibited four distinct phases. These results showed that the E. faecium R8 strain had potential probiotic characteristics and could be used as a candidate strain for aquatic probiotics.

Histochemical distribution of four types of enzymes and mucous cells in the intestine of koi carp (Cyprinus carpio var. koi).

The main purpose of this study was to investigate the distribution of acid phosphatase (ACP), alkaline phosphatase (ALP), non-specific esterase (NSE), peroxidase (POD), and mucous cells in the intestine of the koi carp Cyprinus carpio var. koi. ACP activity was located in the striated border, enterocytes, and lamina propria of the anterior and middle intestines. The ACP activity in the anterior intestine was higher than that in the middle and posterior intestines. ALP existed in the striated border of enterocytes and lamina propria, serosa, muscular layer, and the junction between muscular layer and submucosa layer of the intestine. The ALP activity in the anterior intestine was higher than that in the middle and posterior intestines. NSE activity was localized in the cytoplasm of enterocytes in the whole intestine, and the middle intestine showed the lower NSE activity than the anterior and posterior intestines. POD activity was localized in the blood cells of the lamina propria and cytoplasm of enterocytes in all intestinal segments. The POD activity among the anterior, middle, and posterior intestines was non-significantly different. Alcian blue periodic acid-Schiff histochemical results revealed three types of mucous cells in the intestine. The total number of mucous cells and percentage of type I cells among the anterior, middle, and posterior intestines were non-significantly different. The percentage of the type II cells was the highest in the posterior intestine, while the lowest in the anterior intestine. The percentage of the type III cells was the highest in the anterior intestine, while the lowest in the posterior intestine.

Impact of DNA extraction methods on the observed microbial communities from the intestinal flora of the penaeid shrimp Litopenaeus vannamei.

Two DNA extraction methods, the Zirmil-beating cell disruption method (ZBC) and the QIAamp fast DNA stool mini kit (QIA), were used to extract DNA from the intestinal flora of the penaeid shrimp Litopenaeus vannamei, and their microbial communities were analyzed using 16S rDNA high-throughput sequencing. Results were obtained in terms of the number of reads, alpha diversity indexes, beta diversity indexes and taxonomic composition. The alpha diversity indexes of the community, according to the ZBC method, were higher than those according to the QIA method. Furthermore, results from the three samples using the ZBC method were less consistent than those where the QIA method was used. Further, using the latter method led to substantive clustering. It is suggested that the QIA method is more stable and repeatable than the ZBC method. Although the two extraction methods shared the major abundant microflora based on 16S rDNA high-throughput sequencing, bias associated with diversity analysis indexes and certain species was observed.

Isolation, Identification and Characteristics of Aeromonas veronii From Diseased Crucian Carp (Carassius auratus gibelio).

Aeromonas species often cause disease in farmed fish. In the present study, dominant bacteria were isolated from diseased crucian carp (Carassius auratus gibelio). Based on this, a bacterial isolate was tentatively named CFJY-623. This isolate was identified as Aeromonas veronii based on analysis of its morphological, physiological, and biochemical features, as well as 16S rRNA and gyrB gene sequences. Six virulence genes related to pathogenicity including aerolysin, cytotonic enterotoxins, elastase, glycerophospholipid: cholesterol acyltransferase, lipase, and serine protease were identified in this A. veronii isolate. The median lethal dosage (LD50) of the CFJY-623 isolate for crucian carp was determined as 1.31 × 107 CFU/mL. Artificial experimental infection showed that the CFJY-623 isolate caused considerable histological lesions in the fish, including tissue cell degeneration, necrosis, and inflammatory cell infiltrating. Drug sensitivity testing showed that the isolate was susceptible to aminoglycosides, carbapenemes, and nitrofurans. Exploring its growing features showed that this isolate exhibited a high level of environmental adaptability. These results provided a scientific basis for the identification of A. veronii and treatment for fish infected by this pathogen.

Transcriptome profiles in the spleen of African catfish (Clarias gariepinus) challenged with Aeromonas veronii.

The African catfish, Clarias gariepinus, an important cultured freshwater species in many countries, possess the characteristic of high disease resistance. However, little genomic information for this character of the fish is available up to now. To address the shortfall and to better understand C. gariepinus immune response to pathogen infection at molecular level, C. gariepinus were challenged with potent A. veronii and the high-throughput RNA sequencing (RNA-seq) technology were employed to produce transcriptomes from spleen. In total, an average of 46,073,372 clean reads obtained were de novo assembled into 156,955 unigenes with an average length of 1082 bp. All of unigenes were annotated to seven public databases. Three comparisons were separately conducted between the infected groups at 3 h, 24 h, 48 h post-challenge and control group. A total of 2482 differentially expressed unigenes (DEGs) were identified. Among these, 114 immune-related DEGs were captured, including 88, 42, and 31 genes at 3 h, 24 h and 48 h after infection respectively, for analysis of expression pattern and enrichment. The 114 DEGs displayed four expression patterns by cluster analysis and they were significantly enriched in 38 pathways (q < 0.01) related to the immune or disease, five of which were NF-kappa B, TNF, NLR, TLR and RLR pathways. Finally, the expression levels of twelve selected immune-related DEGs involved in above five pathways were scrutinized. Seven of which were up-regulated at 3 h after infection, afterward, their expression dropped to control level. In summary, this study provides valuable transcriptome resource for understanding the defense mechanisms of C. gariepinus in resistance to pathogens from the gene expression viewpoint, which also open up the possibility to study the immune complexity and to better comprehend the interrelationships between some immune pathways in C. gariepinus.

Biases from different DNA extraction methods in intestine microbiome research based on 16S rDNA sequencing: a case in the koi carp, Cyprinus carpio var. Koi.

This study examined the technical bias associated with different DNA extraction methods used in microbiome research. Three methods were used to extract genomic DNA from the same intestinal microbiota sample that was taken from the koi carp Cyprinus carpio var. koi, after which their microbial diversity and community structure were investigated on the basis of a 16S rDNA high-throughput sequencing analysis. Biased results were observed in relation to the number of reads, alpha diversity indexes and taxonomic composition among the three DNA extraction protocols. A total of 1,381 OTUs from the intestinal bacteria were obtained, with 852, 759, and 698 OTUs acquired, using the Lysozyme and Ultrasonic Lysis method, Zirmil-beating Cell Disruption method, and a QIAamp Fast DNA Stool Mini Kit, respectively. Additionally, 336 OTUs were commonly acquired, using the three methods. The results showed that the alpha diversity indexes (Rarefaction, Shannon, and Chao1) of the community that were determined using the Lysozyme and Ultrasonic Lysis method were higher than those obtained with the Zirmil-beating Cell Disruption method, while the Zirmil method results were higher than those measured, using the QIAamp Fast DNA Stool Mini Kit. Moreover, all the major phyla (ratio>1%) could be identified with all three DNA extraction methods, but the phyla present at a lower abundance (ratio <1%) could not. Similar findings were observed at the genus level. Taken together, these findings indicated that the bias observed in the results about the community structure occurred primarily in OTUs with a lower abundance. The results of this study demonstrate that possible bias exists in community analyses, and researchers should therefore be conservative when drawing conclusions about community structures based on the currently available DNA extraction methods.

Transcriptome profiling of immune-responsive genes in the intestine of Cynoglossus semilaevis Günther challenged with Shewanella algae.

To better understand gene expression in the intestine after Shewanella algae infection and provide insights into its immune roles in the tongue sole, Cynoglossus semilaevis, sequencing-based high-throughput RNA analysis (RNA-Seq) for the intestines between the control group and 12 h post-injection group was performed. After assembly, there was an average of 23,957,159 raw sequencing reads, and 23,943,491 clean reads were obtained after filtering out low-quality reads. Then, 383 differentially expressed genes (DEGs) in the intestines in response to S. algae infection were identified. Subsequently, gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of the DEGs were conducted to further explore their functions. Among all of the pathways involved, sixteen pathways were related to the immune system, among which the complement and coagulation cascades pathway was the most prominent for immunity-related DEGs, followed by the leukocyte transendothelial migration pathway. Furthermore, the expression levels of twelve selected DEGs in the immune-related pathways were identified by quantitative real-time polymerase chain reaction, substantiating the reliability and reproducibility of the RNA-Seq results. In summary, this study represents an important genomic resource for understanding the potential immune role of the tongue sole intestine from the perspective of gene expression.

A modified method for genomic DNA extraction from the fish intestinal microflora.

A modified genomic DNA extraction method named the combination of lysozyme and ultrasonic lysis (CLU) method was used to analyze the fish intestinal microflora. In this method, the physical disruption and chemical lysis steps were combined, and some parameters in the key steps were adjusted. In addition, the results obtained by this method were compared with the results obtained by the Zirmil-beating cell disruption method and the QIAamp Fast DNA Stool Mini Kit. The OD260/OD280 ratio and concentration of the DNA extracted using the CLU method were 2.02 and 282.8 µg/µL, respectively; when the incubation temperatures for lysozyme and RNase were adjusted to 37 °C, those values were 2.08 and 309.8 µg/µL, respectively. On the agarose gel, a major high-intensity, discrete band of more than 10 kb was found for the CLU method. However, the smearing intensity of degraded DNA was lower when the incubation temperatures were 60 °C for lysozyme and 30 °C for RNase than when incubation temperatures of 37 °C for lysozyme and 37 °C for RNase were used. The V3 variable region of the prokaryotic 16S rDNA was amplified, and an approximately 600-bp fragment was observed when the DNA extracted using the CLU method was used as a template. The CLU method is simple and cost effective, and it yields high-quality, unsheared, high-molecular-weight DNA, which is comparable to that obtained with a commercially available kit. The extracted DNA has potential for applications in critical molecular biology techniques.

First sporadic case of pathogenic Escherichia coli infection in Black swan in China.

A strain of bacteria was isolated from the diseased black swan (Cygnus atratus) died from enteritis diarrhea, and designated tentatively as B-1 strain. Morphological and biochemical tests, as well as phylogenetic analysis derived from 16S rRNA and fimC gene sequencing both strongly indicated that B-1 strain is identical to Escherichia coli. Furthermore, the polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) profile of the isolate was different from that of two reference strains. Antibiotic sensitivity testing of B-1 strain was carried out by the standard Kirby-Bauer disc diffusion method. Animal experiments demonstrated that B-1 strain is pathogenic to mice and chickens. This is first sporadic case of pathogenic E. coli infection in Black swan in China.

Nocardia rhizosphaerae sp. nov., a novel actinomycete isolated from the coastal rhizosphere of Artemisia Linn., China.

A novel actinomycete, designated strain KLBMP S0043(T), was isolated from the rhizosphere soil of Artemisia Linn. collected from the coastal region of Lianyungang, Jiangsu Province, in east China and was studied in detail for its taxonomic position. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain KLBMP S0043(T) is a member of the genus Nocardia. The 16S rRNA gene sequence similarity indicated that strain KLBMP S0043(T) is closely related to Nocardia asteroides NBRC 15531(T) (97.61 %) and Nocardia neocaledoniensis SBHR OA6(T) (97.38 %); similarity to other type strains of the genus Nocardia was found to be less than 97.2 %. The organism has chemical and morphological features consistent with its classification in the genus Nocardia such as meso-diaminopimelic acid as the diagnostic diamino acid in the cell wall peptidoglycan and arabinose and galactose as the diagnostic sugars. The predominant menaquinone was identified as MK-8(H4ω-cycl). Mycolic acids were detected. The diagnostic phospholipids were found to be diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides. The predominant cellular fatty acids were identified as C16:0, C18:0, C18:1ω9c, 10-methyl C18:0 [tuberculostearic acid (TBSA)] and summed feature 3 (C16:1ω7c/C16:1ω6c). The G+C content of the genomic DNA was determined to be 71.4 mol%. The results of DNA-DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of the strain from its most closely related strains. Based on morphological, chemotaxonomic and phylogenetic data, strain KLBMP S0043(T) is considered to represent a novel species of the genus Nocardia, for which the name Nocardia rhizosphaerae sp. nov. is proposed. The type strain is KLBMP S0043(T) (=CGMCC 4.7204 (T) = KCTC 29678(T)).