Hua Yue

Unexpected sequence types in livestock associated methicillin-resistant Staphylococcus aureus (MRSA): MRSA ST9 and a single locus variant of ST9 in pig farming in China

In October 2008 nine farrow-to-finish pig farms were visited in Shuangliu County in Sichuan Province, China. One farm was empty for one month but not cleaned after depopulation. Dust samples were collected at each farm and analysed for the presence of methicillin-resistant Staphylococcus aureus (MRSA). Dust samples from four farms were also analysed for the presence of methicillin-susceptible S. aureus (MSSA). On 5/9 farms MRSA was isolated and on 2/4 farms MSSA was isolated. On two farms, including the empty farm, no MRSA or MSSA could be detected. All MRSA isolates (n=43) belonged to spa type t899. MSSA isolates belonged to spa type t899 (n=12) and spa type t034 (n=2). From 4/9 farms the MRSA isolates of spa type t899 were assigned to multilocus sequence type (MLST) ST9 whereas on one farm the MRSA spa type t899 isolates belonged to a single locus variant of MLST ST9 (ST1376). MSSA isolates with spa type t899 belonged to MLST ST9 and the MSSA with spa type t034 belonged to MLST ST398. This is the first report on MRSA in pig farms in China and the first time that MRSA ST9 and a single locus variant of ST9 are detected in pig farms. This study shows that livestock associated MRSA is not restricted to clonal lineage ST398 as found in Europe and Northern America in commercial pigs but that other MRSA lineages are able to spread in livestock as well. The study confirms that livestock may act as a reservoir for MRSA.

Viral metagenomics analysis demonstrates the diversity of viral flora in piglet diarrhoeic faeces in China.

To investigate the diversity of viral flora, we used metagenomics to study the viral communities in a pooled faecal sample of 27 diarrhoeic piglets from intensive commercial farms in China. The 15 distinct mammalian viruses identified in the pooled diarrhoeic sample were, in order of abundance of nucleic acid sequence, Porcine epidemic diarrhea virus (PEDV), sapovirus, porcine bocavirus-4 (PBoV-4), sapelovirus, torovirus, coronavirus, PBoV-2, stool-associated single-stranded DNA virus (poSCV), astrovirus (AstV), kobuvirus, posavirus-1, porcine enterovirus-9 (PEV-9), porcine circovirus-like (po-circo-like) virus, picobirnavirus (PBV) and Torque teno sus virus 2 (TTSuV-2). The prevalence rate of each virus was verified from diarrhoeic and healthy piglets by PCR assay. A mean of 5.5 different viruses were shed in diarrhoeic piglets, and one piglet was in fact co-infected with 11 different viruses. By contrast, healthy piglets shed a mean of 3.2 different viruses. Compared with samples from healthy piglets, the co-infection of PEDV and PBoV had a high prevalence rate in diarrhoea samples, suggesting a correlation with the appearance of diarrhoea in piglets. Furthermore, we report here for the first time the presence of several recently described viruses in China, and the identification of novel genotypes. Therefore, our investigation results provide an unbiased survey of viral communities and prevalence in faecal samples of piglets.

Reference gene selection for normalization of PCR analysis in chicken embryo fibroblast infected with H5N1 AIV

Chicken embryo fibroblasts (CEFs) are among the most commonly used cells for the study of interactions between chicken hosts and H5N1 avian influenza virus (AIV). In this study, the expression of eleven housekeeping genes typically used for the normalization of quantitative real-time PCR (QPCR) analysis in mammals were compared in CEFs infected with H5N1 AIV to determine the most reliable reference genes in this system. CEFs cultured from 10-day-old SPF chicken embryos were infected with 100 TCID50 of H5N1 AIV and harvested at 3, 12, 24 and 30 hours post-infection. The expression levels of the eleven reference genes in infected and uninfected CEFs were determined by real-time PCR. Based on expression stability and expression levels, our data suggest that the ribosomal protein L4 (RPL4) and tyrosine 3-monooxygenase tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ) are the best reference genes to use in the study of host cell response to H5N1 AIV infection. However, for the study of replication levels of H5N1 AIV in CEFs, the β-actin gene (ACTB) and the ribosomal protein L4 (RPL4) gene are the best references.

Selection of reference genes for quantitative real-time PCR analysis in chicken embryo fibroblasts infected with avian leukosis virus subgroup J

BackgroundThe selection of stably expressed reference genes is a prerequisite when evaluating gene expression, via real-time PCR, in cells in response to viral infections. The objective of our study was to identify suitable reference genes for mRNA expression analysis in chicken embryonic fibroblasts (CEF) after infection with avian leukosis virus subgroup J (ALV-J).FindingsThe expression levels of 11 potential reference genes in CEF infected with ALV-J were determined by real-time PCR. The expression stability of these genes were analyzed and ranked using the geNorm tool. Analysis indicated that the genes RPL30 (ribosomal protein L30) and SDHA (succinate dehydrogenase complex, subunit A) were the most stably expressed genes in the ALV-J infected CEF.ConclusionsThe RPL30 and SDHA were deemed suitable for use as reference genes for real-time PCR analysis of mRNA gene expression during ALV-J infection, whereas commonly used ACTB and GAPDH are unsuitable to be reference genes.

Phenotypic characterization and prevalence of enterotoxin genes in Staphylococcus aureus isolates from outbreaks of illness in Chengdu City.

Staphylococcus aureus produces a spectrum of enterotoxin that is recognized as the main reason for causing staphylococcal food poisoning. The aim of the current study was to investigate the phenotypic characteristics and enterotoxin genotypes of S. aureus isolated from food poisoning sufferers. On the basis of the amplification of 16S rRNA and nuc gene specific to S. aureus assay and the phenotype (hemolytic activity, thermal stable nuclease [Tnase] test, and biofilm formation), all isolates were identified as S. aureus. To genotypically characterize S. aureus isolates, genes encoding staphylococcal enterotoxin (sea, seb, sec, sed, see, seg, seh, sei, sej, sek, sem, sen, ser, and seu) were investigated by using polymerase chain reaction technique. The results showed that the eight isolates of S. aureus had different enterotoxin genotypic characteristics, which was the main cause of food poisoning. One isolate contained 10 enterotoxin genes, and the other 7 isolates carried 3 or more enterotoxin genes. The frequency of the newly identified enterotoxin genes (seg-seu) was higher than classical genes (sea-see). Overall, multi-gene detection rates were 75% (for sek, ser, and seu); 50% (for sea and sem); 37.5% (for sen, seg, and sei); and 12.5% (for seb, sec, sed, and sej), respectively. The see and seh gene were not detected in any isolates. The current study provided the exact distribution of enterotoxin genes in eight S. aureus strains from food poisoning sufferers, which indicated that the pathogenicity of the newly identified enterotoxin should be highlighted. The need for prevention of food poisoning occurrences caused by enterotoxin of S. aureus should be reinforced.

Development of Quantitative Real-Time Polymerase Chain Reaction for Duck Enteritis Virus DNA

Abstract Duck enteritis virus (DEV) is a herpesvirus that causes an acute, contagious, and fatal disease. In the present article, we introduce a quantitative real-time polymerase chain reaction (PCR) assay for DEV DNA using TaqMan technology and a two-step protocol. It was confirmed to be rapid, sensitive, and specific for DEV detection. The primers and probe were designed and directed to the DNA polymerase gene of DEV. The method will provide a valuable tool for rapid laboratory diagnosis of DEV infection. By virtue of its high-throughput format and its ability to accurately quantify the viral DNA, the method may be useful for large epidemiological surveys and clarification of pathogenesis, such as latency and reactivation of the virus.

Update on the pathogenesis of Haemophilus parasuis infection and virulence factors.

Haemophilus parasuis is the causative agent of Glässers disease in pigs, a severe systemic disease that has led to increasing economic losses in the pig industry worldwide. The H. parasuis genome sequence has been completed, but the function and essentiality of the annotated genes remain largely unknown, especially virulence factors. The recent developments in the efficient genetic manipulation of H. parasuis have greatly facilitated the study of gene function, pathogenesis mechanisms and virulence factors. In this review, we provided update information regarding that (i) how the pathogen overcome host immune responses and cell barriers which were tightly associated with the pathogenesis, and (ii) the several recent identification of virulence factors were involved in evading the immune responses and cell barriers in H. parasuis.

Transcriptome analysis of duck liver and identification of differentially expressed transcripts in response to duck hepatitis A virus genotype C infection.

Background Duck is an economically important poultry and animal model for human viral hepatitis B. However, the molecular mechanisms underlying host–virus interaction remain unclear because of limited information on the duck genome. This study aims to characterize the duck normal liver transcriptome and to identify the differentially expressed transcripts at 24 h after duck hepatitis A virus genotype C (DHAV-C) infection using Illumina–Solexa sequencing. Results After removal of low-quality sequences and assembly, a total of 52,757 unigenes was obtained from the normal liver group. Further blast analysis showed that 18,918 unigenes successfully matched the known genes in the database. GO analysis revealed that 25,116 unigenes took part in 61 categories of biological processes, cellular components, and molecular functions. Among the 25 clusters of orthologous group categories (COG), the cluster for “General function prediction only” represented the largest group, followed by “Transcription” and “Replication, recombination, and repair.” KEGG analysis showed that 17,628 unigenes were involved in 301 pathways. Through comparison of normal and infected transcriptome data, we identified 20 significantly differentially expressed unigenes, which were further confirmed by real-time polymerase chain reaction. Of the 20 unigenes, nine matched the known genes in the database, including three up-regulated genes (virus replicase polyprotein, LRRC3B, and PCK1) and six down-regulated genes (CRP, AICL-like 2, L1CAM, CYB26A1, CHAC1, and ADAM32). The remaining 11 novel unigenes that did not match any known genes in the database may provide a basis for the discovery of new transcripts associated with infection. Conclusion This study provided a gene expression pattern for normal duck liver and for the previously unrecognized changes in gene transcription that are altered during DHAV-C infection. Our data revealed useful information for future studies on the duck genome and provided new insights into the molecular mechanism of host–DHAV-C interaction.

Genome Sequence of Mycoplasma ovipneumoniae Strain SC01

Mycoplasma ovipneumoniae is associated with chronic nonprogressive pneumonia in both sheep and goats. Studies concerning its molecular pathogenesis, genetic analysis, and vaccine development have been hindered due to limited genomic information. Here, we announce the first complete genome sequence of this organism.

Analysis of gene expression profiles reveals the regulatory network of cold-inducible RNA-binding protein mediating the growth of BHK-21 cells

Cold‐inducible RNA‐binding protein (Cirp), the first cold‐shock protein identified in mammals, is a sensor protein whose expression increases in response to stress. Recent reports have shown that Cirp is involved in cell proliferation, development, circadian modulation under physiological conditions, and tumor formation and progression. However, the molecular mechanisms underlying the activities of Cirp in the mammalian kidney cells remain unclear. In this study, we constructed BHK‐21cells overexpressing Cirp (Cirp + BHK‐21) knockdown BHK‐21 cells (Cirp − BHK‐21) to investigate the function of Cirp in cell proliferation. We analyzed the gene expression of Cirp − BHK‐21 cells using genome‐wide expression microarrays to explore the molecular mechanism of Cirp action. We found that (1) Cirp overexpression significantly enhanced cell proliferation, whereas Cirp knockdown dramatically reduced cell proliferation, suggesting that Cirp is a positive regulator of BHK‐21 cell proliferation. (2) Differentially expressed genes in Cirp − BHK‐21 and control cells were shown to be involved in many biological processes. (3) Pathway analysis showed that five enriched pathways, namely, Focal adhesion, Mapk, Wnt, Apoptosis, and Cancer‐related signaling pathways, were identified as central pathway networks regulated by Cirp. These results can provide new insights into the molecular mechanisms of Cirp function.