Detection of mcr-1-harbouring Escherichia coli by quantum dot labelling of synthetic small peptides mimicking lipopolysaccharide receptors.

Overuse of antibiotics and the emergence of multidrug-resistant bacteria has made colistin the last line of defence against complex infections. In previous studies, MCR-1-mediated colistin resistance was mainly detected through PCR or antimicrobial susceptibility testing. However, intuitive detection methods for phenotype are rarely reported. In this study, two small peptide antibodies were constructed for immunofluorescence detection of mcr-1-harbouring Escherichia coli: one was a small peptide labelled with a quantum dot antibody; and the other was a small peptide labelled with a fluorescein isothiocyanate (FITC) antibody. Whether using FITC or quantum dots, colistin-resistant bacteria in the sample could be qualitatively detected. The assembled antibodies achieved the desired goals in terms of sensitivity, specificity, precision and repeatability. The non-specific problem of sandwich antigen recognition of lipid A binding to small peptides in modified lipopolysaccharide (LPS) was resolved, and this relatively developed immunofluorescence technique standardised the detection process. Together, in addition to PCR, both fluorescent antibodies can be used for immunofluorescent detection of mcr-1-harbouring E. coli.

Integrated expression profiles of mRNA and miRNA in a gerbil model of fatty liver fibrosis treated with exenatide.

BACKGROUND: The morbidity of nonalcoholic fatty liver disease (NAFLD) has increased consistently in recent years. Exenatide could reverse liver fibrosis and lower the occurrence of fatty liver. The aim of the study was to identify and characterize mRNA and miRNA expression to elucidate the mechanism of exenatide in the gerbil model. METHODS: Gerbils were fed a high-fat diet for 8 weeks to induce a fibrosis model; then, the gerbil models were treated with exenatide for 4 weeks. The total RNA extracted from the liver tissue samples was used to prepare the library and sequence on a HiSeq 2000. Bioinformatic methods were employed to analyze the sequence data to identify the mRNAs and miRNAs and to acquire the miRNA-mRNA regulatory network. RESULTS: By RNA-seq, 2344 differentially expressed genes (DEGs) and 72 miRNAs were found in the model group. Compared with the model group, 591 DEGs and 19 miRNAs were found in the quercetin group, whereas 876 DEGs and 18 miRNAs were found in the treatment group. The miRNA-mRNA regulatory network was constructed in a gerbil model. Immunohistochemistry and RNA sequencing confirmed that the therapeutic effect of exenatide may be derived from extrahepatic signal transduction. The key differential genes are CYP3A, CYP4A11, ACAA1, ACSM, PHX1, MAO, FMO, UGT, ACOX2, ABAT, PIK3C and PLCG1. The key miRNAs are miR-15a, miR-27b, miR-532-3P, miR-627, miR-3596, miR-142-3P, Let-7e-5p, miR-214-5, miR-101-3p, miR-378d. New miRNAs, such as novel_127, novel_143, novel_15, novel_204 are associated with liver fibrosis, while novel_127, novel_15, and novel_54 are associated with reverse treated with exenatide. CONCLUSIONS: Our research represents the first description of mRNA/miRNA profiles in a gerbil model of fatty liver fibrosis treated with exenatide, which may provide insights into the pathogenesis or treatment of the metabolic syndrome.

Immunogenicity and protective efficacy of an EB66® cell culture-derived duck Tembusu virus vaccine.

The avian EB66® cell line, derived from duck embryonic stem cells, has been widely used for producing human and animal therapeutic proteins and vaccines. In current study we evaluated the potential use of EB66® cell line in a cell culture-derived duck Tembusu virus (DTMUV) vaccine development. After optimizing the growth conditions of DTMUV HB strain in EB66® cells, we successfully generated three batches of viruses with ELD50 titres of 105.9/0.1 ml, 105.3/0.1 ml and 105.5/0.1 ml, respectively, for using in the preparation of inactivated vaccines. The immunogenicity and protective efficacy of these EB66® cells-derived inactivated vaccines were examined in ducks. Results indicated that all three batches of vaccines induced haemagglutination-inhibition (HI) antibody response in immunized birds at 2 weeks after a single immunization. Immunized ducks and ducklings were protected against a virulent challenge at 4 weeks after a booster immunization. The duration of immunity was for 3-4 months after a booster immunization. These results demonstrated the feasibility of using EB66® cell line to grow up DTMUV for vaccine preparation. RESEARCH HIGHLIGHTS Duck Tembusu virus can be propagated in EB66® cells. EB66® cell-derived inactivated DTMUV vaccines are immunogenic and can provide protection against a virulent challenge. A long-lasting immunity is induced after a booster immunization.

Development of a Hemagglutination Inhibition Assay for Duck Tembusu Virus.

The HB strain of duck Tembusu virus (DTMUV) propagated in the brains of newborn mice was used to prepare antigens for use in the hemagglutination inhibition (HI) test. Results showed that such prepared antigens are highly specific to the serum samples derived from DTMUV-infected animals. No spurious hemagglutination reactions against serum samples specific to avian influenza virus H5, H7, H9 subtypes, Newcastle disease virus, egg drop syndrome virus, duck plague virus, and duck hepatitis A virus were observed. The HI test can detect specific antibodies in the serum samples as early as day 4 after experimental infection of ducks with DTMUV. When compared to a virus neutralization test, the sensitivity is 100%. Overall, the HI test developed is highly specific to DTMUV and can be used in clinical diagnosis of diseases and in vaccine studies to monitor the kinetics of antibody response.

Desarrollo de un ensayo de inhibición de la hemaglutinación para el virus Tembusu del pato. La cepa HB del virus Tembusu del pato (DTMUV) propagada en el cerebro de ratones recién nacidos se usó para preparar antígenos para su uso en la prueba de inhibición de la hemaglutinación (HI). Los resultados mostraron que tales antígenos preparados son altamente específicos para las muestras de suero derivadas de animales infectados con el virus Tembusu del pato. No se observaron reacciones inespecíficas de hemaglutinación con muestras de suero específicas para el virus de la influenza aviar subtipos H5, H7 y H9, virus de la enfermedad de Newcastle, virus del síndrome de baja de postura, virus de la enteritis viral del pato y virus de la hepatitis A del pato. La prueba de inhibición de la hemaglutinación puede detectar anticuerpos específicos en las muestras de suero desde el día cuatro después de la infección experimental de patos con el virus Tembusu. Cuando se comparó con una prueba de neutralización viral, la sensibilidad es del 100%. En general, la prueba de inhibición de la hemaglutinación desarrollada es altamente específica para el virus Tembusu y se puede utilizar en el diagnóstico clínico de enfermedades y en estudios de vacunas para controlar la cinética de la respuesta de anticuerpos.

Pigeons are resistant to experimental infection with H7N9 avian influenza virus.

To determine the susceptibility of pigeons to the newly emerged avian influenza virus subtype H7N9, we experimentally infected three different types of pigeons (meat, town, and racing) with two different doses (2 × 10(4) or 2 × 10(5) EID50) of H7N9 avian influenza virus A/Chicken/China/2013 by either intranasal and intraocular inoculation (IN + IO) or intravenous injection (IV). In addition, the potential transmission of H7N9 to pigeons by direct close contact with experimentally infected pigeons and chickens was assessed. Results showed that none of the experimentally infected pigeons exhibited any clinical signs regardless of the infection route and dose. Of the 12 racing pigeons that were randomly selected and necropsied, none of them had any gross lesions. In agreement with this finding, virus was not isolated from all pigeons. No detectable H7-specific antibodies were found in any pigeon. In contrast, 11 of 31 chickens that were either directly infected with H7N9 by IN + IO inoculation or by contact with IN + IO-infected chickens had conjunctivitis. Virus was isolated from all 31 chickens and H7-specific antibodies were detected in these chickens. However, none of the IV-infected chickens or chickens in direct contact with IV-infected chickens had any clinical signs. No virus was isolated from these chickens and no H7-specific antibody was detected. Overall, we conclude that pigeons are less or not susceptible to the H7N9 virus at the doses used and are not likely to serve as a reservoir for the virus. However, the virus does cause conjunctivitis in chickens and can transmit to susceptible hosts by direct contact.

Efficacy Evaluation of an Inactivated Duck Tembusu Virus Vaccine.

To evaluate the potential use of an inactivated virus-based vaccine for the control and prevention of the newly emerged duck Tembusu virus infection in China, a duck Tembusu virus isolate, Tembusu-HB, was propagated in 12-day-old duck embryos and inactivated by treatment with formaldehyde. The inactivated viral antigen was emulsified with mineral oil, and five batches of the vaccine were manufactured. The immunogenicity and protection efficacy of the vaccine were evaluated in Beijing ducks and Beijing white geese. Results showed that more than 80% of immunized ducks were protected against virulent virus challenge after two intramuscular or subcutaneous injections of the inactivated vaccine, as evidenced by the negative virus isolation results. The protection is also correlated with a positive virus-specific antibody response as detected by ELISA. In contrast, none of the control ducks and geese had any detectable antibody response. Virus was isolated from all control ducks and geese after virulent virus challenge. Interestingly, a variable level of protection (20%-80%) was observed in Beijing white geese immunized twice with the same batches of vaccine, suggesting a species-specific effect of the vaccine. Overall, the results clearly suggest that the inactivated duck Tembusu virus vaccine is immunogenic and provides protection against virulent virus challenge.

Primary genetic investigation of a hyperlipidemia model: molecular characteristics and variants of the apolipoprotein E gene in Mongolian gerbil.

The objective of this work was to establish a novel Mongolian gerbil (Meriones unguiculatus) hyperlipidemia model and to investigate its susceptibility genetic basis. Two rodent (gerbil and rat) hyperlipidemia models were induced by feeding a high fat/high-cholesterol (HF/HC) diet. There were significant increases of serum total cholesterol, triglycerides, low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in gerbils within a 4-week modeling period. About 10-30% of >8-month-old individuals developed hyperlipidemia spontaneously. The apolipoprotein E (ApoE) gene was cloned by merging a sequence of rapid amplification of cDNA ends (RACE) and nested polymerase chain reaction products. The results revealed an open reading frame of 948 bp, encoding a protein of 298 amino acids. The gene without a 5'-UTR region in the first intron was highly homologous to human Apo-A-I and rat Apo-A-IV. The distribution of expression of the ApoE gene in liver, brain, heart, lung, kidney, and adrenal gland was detected by an ABC immunohistochemical procedure. Three single nucleotide polymorphisms (SNPs; C97T, G781T, and A1774T) were first found using PCR-single-strand conformation polymorphism (PCR-SSCP) in a closed population containing 444 animals. Correlation analysis confirmed that new SNPs, age, and gender were associated significantly (P < 0.05) with hyperlipidemia.

Hypolipidemic and antioxidant activities of polysaccharides from Rosae Laevigatae Fructus in rats.

Two major fractions (RLP-1 and RLP-2) were obtained by purifying the crude polysaccharides extracted from a traditional Chinese herb Rosae Laevigatae Fructus. The average molecular weight of RLP-1 and RLP-2 was 21.5 kDa and 16.1 kDa, respectively. Monosaccharide analysis indicated that RLP-1 was composed of xylose, mannose and galactose in the molar ratio of 1:11:8, while RLP-2 was only a glucan. Oral administration of RLP-1 could significantly decrease levels of serum total cholesterol (TC), triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C), inhibit hepatic lipid accumulation, increase antioxidant lipids and up-regulate expressions of peroxisome proliferator-activated receptor-γ (PPAR-γ) and lipoprotein lipase (LPL) in hyperlipidemia rats. These results suggest that RLP-1 improve hyperlipidemia possibly through regulating PPAR-mediated lipid metabolism. Therefore, could be explored as a possible agent for hyperlipidemia.

Dietary quercetin ameliorates nonalcoholic steatohepatitis induced by a high-fat diet in gerbils.

Dietary quercetin is highly abundant in edible plants, which possesses a wide range of pharmacological properties. This study was to investigate hepatoprotective effects of quercetin in the nonalcoholic steatohepatitis (NASH) gerbils induced by a high-fat diet (HFD), and to evaluate its regulatory mechanism on hepatic inflammatory response. The gerbils were fed with HFD for 28 days to induce NASH. From 15th day to 28th day, the treated drugs were given daily to each animal, respectively. The lipid profiles and biochemical markers were determined at the end of the experiment. The expressions of Sirt1, NF-κB p65 and iNOS were detected by immunohistochemistry and Western blot analysis. The results showed that oral administration of quercetin at doses of 30-60 mg/kg to hyperlipidemia rats for 14 days were highly effective in decreasing the levels of serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), alanine aminotransferase (ALT) and aspartate aminotransferase (AST). It could decrease lipid accumulation in the hepatocytes, and reduce serum levels of pro-inflammatory cytokines TNF-α and IL-6 via regulating the expressions of Sirt1, NF-κB p65 and iNOS. Thus, dietary quercetin had significant therapeutic benefits and could be explored as a potential promising candidate for the prevention of NASH.

[Transdermal behavior of effective constituents in xuanbi gel plaster].

OBJECTIVE: To study the transdermal absorbability of gentiopicroside, naringin and protosappanin B contained in Xuanbi gel plaster. METHOD: The Franz diffusing cells method was adopted for the in vitro model of rat belly skins. Three indexes, gentiopicroside, naringin and protosappanin B, residued in the accept liquid, skins and plaster were determined by HPLC. RESULT: The penetration rates of gentiopicroside, naringin and protosappanin B were respectively 3.47, 1.59, 2.13 microg x cm(-2) x h(-1). After 24 h, their penetration rates were 25.42%, 11.73%, 17.78%, respectively. The residual quantities of gentiopieroside, naringin and protosappanin B in skin were 0.231, 0.593, 0.568 microg x cm(-2), ith the retention rates of 0.027%, 0.227%, 0.475%, respectively. The amount of residue of gentiopicroside, naringin and protosappanin B in plaster were 2179, 674, 278 microg, with the retention rates of 81.36%, 81.92%, 73.83%, respectively. CONCLUSION: The in vitro transdermal behavior of Xuanbi gel plaster is close to a zero-order process. The residual quantity the retention rate in skins is much lower than the penetration rate and the residual rate in plaster.

A survey of avian influenza in tree sparrows in China in 2011.

Tree sparrows (Passer montanus) are widely distributed in all seasons in many countries. In this study, a survey and relevant experiments on avian influenza (AI) in tree sparrows were conducted. The results suggested that the receptor for avian influenza viruses (AIVs), SAα2,3Gal, is abundant in the respiratory tract of tree sparrows, and most of the tree sparrows infected experimentally with two H5 subtype highly pathogenic avian influenza (HPAI) viruses died within five days after inoculation. Furthermore, no AIVs were isolated from the rectum eluate of 1300 tree sparrows, but 94 serological positives of AI were found in 800 tree sparrows. The serological positives were more prevalent for H5 subtype HPAI (94/800) than for H7 subtype AI (0/800), more prevalent for clade 2.3.2.1 H5 subtype HPAI (89/800) than for clade 2.3.4 (1/800) and clade 7.2 (4/800) H5 subtype HPAI, more prevalent for clade 2.3.2.1 H5 subtype HPAI in a city in southern China (82/800) than in a city in northern China (8/800). The serological data are all consistent with the distribution of the subtypes or clades of AI in poultry in China. Previously, sparrows or other passerine birds were often found to be pathogenically negative for AIVs, except when an AIV was circulating in the local poultry, or the tested passerine birds were from a region near waterfowl-rich bodies of water. Taken together, the data suggest that tree sparrows are susceptible to infection of AIVs, and surveys targeting sparrows can provide good serological data about the circulation of AIVs in relevant regions.

Tembusu virus in ducks, china.

In China in 2010, a disease outbreak in egg-laying ducks was associated with a flavivirus. The virus was isolated and partially sequenced. The isolate exhibited 87%-91% identity with strains of Tembusu virus, a mosquito-borne flavivirus of the Ntaya virus group. These findings demonstrate emergence of Tembusu virus in ducks.

[Molecular genetic evolution analysis of new A(H1N1) influenza virus].

In order to analyze the molecular epidemiology of A (H1N1) influenza virus in 2009, the complete genome sequences of influenza strains from different host sources downloaded from the NCBI were analyzed on genetic evolution by DNAstar software in this research. The results showed that 79 mutation sites of new A (H1N1) influenza virus were observed compared to previous human A (H1N1) influenza strain, including 14 mutation sites new in all A (H1N1) influenza sources and 37 mutation sites only observed in swine strain. A significant difference was represented in antigenic sites between new A (H1N1) influenza strain and the previous human A (H1N1) strain. This phenomenon shows the new A (H1N1) influenza strain is either originated from the recombination of human and swine strain or from the infection in pig populations and gradual mutation to human tansmission, which remains to be further studied.

Influenza A virus receptors in the respiratory and intestinal tracts of pigeons.

To understand the basis of the resistance of pigeons to Asian lineage highly pathogenic avian influenza virus (HPAIV) subtype H5N1, we examined the presence of influenza virus receptors, sialic acids linked to galactose by an alpha-2,3 linkage (SAalpha2,3Gal) or by an alpha-2,6 linkage (SAalpha2,6Gal), in respiratory and intestinal tracts of pigeons and compared the distributions of N-acetylneuraminic and N-glycolylneuraminic acids in the trachea and intestines of pigeons and chickens. Results suggested that the epithelial surfaces of the larynx, trachea, bronchus, and bronchiole of pigeons contained abundant SAalpha2,6Gal with little or no SAalpha2,3Gal. In contrast, the epithelial surfaces of the pharynx, trachea, bronchus, and bronchiole of chickens contained mainly SAalpha2,3Gal, a well-recognized receptor for avian influenza viruses including H5N1 HPAIV. A similar distribution pattern of N-acetylneuraminic and N-glycolylneuraminic acids in the trachea and intestines of pigeons and chickens was observed. Overall, the results suggest that SAalpha2,6Gal is the major receptor in the pigeon airway, which may partly contribute to the resistance of pigeons to Asian lineage HPAIV subtype H5N1.

Susceptibility and transmissibility of pigeons to Asian lineage highly pathogenic avian influenza virus subtype H5N1.

To determine whether or not pigeons are susceptible to infection with Asian lineage highly pathogenic (HP) avian influenza virus (AIV) subtype H5N1 and can serve as a transmission host for H5N1 HPAIV, we experimentally infected 187 young and adult pigeons with five different isolates of H5N1 HPAIV and co-habited some experimentally infected pigeons with susceptible specific pathogen free chickens. Results showed that all infected pigeons remained clinically healthy during the observation period. No gross lesions or histopathological changes were observed in the infected pigeons, and haemagglutination inhibition antibodies were not detected in serum samples of the infected pigeons. Additionally, all chickens placed in contact with AIV H5N1 infected pigeons remained healthy, and no virus or haemagglutination inhibition antibodies were detected in samples from the chickens. Our data suggest that pigeons are not susceptible to Asian lineage H5N1 HPAIV and do not transmit the virus to chickens.