Loop-mediated isothermal amplification assay targeting the omp25 gene for rapid detection of Brucella spp.

A novel loop-mediated isothermal amplification (LAMP) assay was established to detect Brucella species DNA in milk and blood samples of animals and humans. This LAMP assay based on the sequence of highly repetitive omp25 gene was able to detect 9fg/µl Brucella spp. DNA with high sensitivity, which was 10 times higher than the nested PCR. The LAMP was evaluated for its specificity using 19 strains of six Brucella species and 28 related non-Brucella micro-organism strains as controls. The target 19 Brucella strains were all amplified, and no cross-reaction was found with all the non-Brucella micro-organism strains. Both nested PCR and LAMP assays were then used to detect Brucella spp. DNA in 78 milk samples and 113 blood samples from animals and 11 blood samples from humans, and the established LAMP assay yielded 99.0% concordance rate with the nested PCR. The LAMP assay should be a potential tool with high convenience, rapidity, sensitivity and specificity for the diagnosis of Brucellosis.

Potential effects of hepatitis E virus infection in swine on public health in China.

Hepatitis E virus (HEV), a zoonotic pathogen, is the main cause of acute hepatitis worldwide. Swine serves as the main reservoir, and its infection is mainly transmitted via fecal-oral route. Due to huge consumption of pork in China, close human-swine interactions at pig farms likely contribute to high risk in zoonotic transmission of HEV. Thus, we aim to investigate the HEV prevalence in pig farm in seven provinces across the east to west China and estimate the potential effects of swine HEV on public health in China. In this study, serum samples of pig were collected for detection of anti-HEV antibodies from the seven provinces. A high seroprevalence of 67.1% was found, and no clear difference was observed among these regions. However, the age and the breeding purpose (for meat supplier or breeding offspring) play significant roles in the risk of swine HEV infection. In addition, sequence comparison of various HEV genomes isolated in China displayed that swine HEV posed obvious threats to ruminant breeding and public health. The high level of seroprevalence of swine HEV strongly plays an important role in cross-species of HEV infection. Therefore, effective measures should be performed to prevent HEV infection from infected pigs to human and other ruminants.

Proteomic analysis of murine bone marrow derived dendritic cells in response to peste des petits ruminants virus.

Peste des petits ruminants virus (PPRV) poses a great threat to livestock husbandry, especially goat farming due to its high mortality and morbidity. Dendritic cells (DCs), as the principal stimulators of naive Th cells were widely used in antigen processing and presenting. In the previous study, we tested the effects of PPRV on murine bone marrow derived dendritic cells (BMDCs) including surface markers and cytokines. While the aim of this study is to detect the proteomic profile of BMDCs stimulated with PPRV towards key proteins involved in. Following PPRV stimulation, 110 differentially expressed proteins (DEPs) were identified through iTRAQ labelling with LC-MS/MS approach, of which 94 DEPs were up-regulated and 16 DEPs were down-regulated, respectively. Among them 15 out of 110 DGPs were related to innate immune system, three were involved in cell apoptosis, RPS15a and Smox were related to translation of viral mRNA. Additionally, western blot analysis showed identical results to iTRAQ analysis. There will be profound significance for understanding antigen-presenting of BMDCs after stimulation with PPRV.

Whole-Genome Sequencing of a Brucella melitensis Strain (BMWS93) Isolated from a Bank Clerk and Exhibiting Complete Resistance to Rifampin.

Human brucellosis has become the most severe public health problem in the Ulanqab region of Inner Mongolia, China. Brucella melitensis BMWS93 was obtained from a blood sample taken from a bank clerk in the Ulanqab region of Inner Mongolia, China, and antimicrobial susceptibility testing in vitro showed no zone of inhibition, which confirmed resistance to rifampin. Therefore, whole-genome sequencing of this isolate was performed to better understand the mechanism of this resistance.

Analyses of nucleotide, synonymous codon and amino acid usages at gene levels of Brucella melitensis strain QY1.

Brucella melitensis is the causative pathogen of the zoonotic disease brucellosis in China. This work focused on analyses of genetic features represented by nucleotide, synonymous codon and amino acid usages at gene levels of B. melitensis strain QY1 isolated from China. Although nucleotide usage biases at different codon positions all work on synonymous codon usage bias, nucleotide usage biases at the 1st and 3rd positions play more important roles in codon usages. Mutation pressure caused by nucleotide composition constraint influences the formation of over-representative synonymous codons, but neighboring nucleotides surrounding a codon strongly influence synonymous codon usage bias for B. melitensis strain QY1. There is significant correlation between amino acid usage bias and hydropathicity of proteins for B. melitensis strain QY1. Compared with different Brucella species about synonymous codon usage patterns, synonymous codon usages are not obviously influenced by hosts. Due to nucleotide usage bias at the 1st codon position influencing synonymous codon and amino acid usages, good interactions among nucleotide, synonymous codon and amino acid usages exist in the evolutionary process of B. melitensis.

The effects of codon usage on the formation of secondary structures of nucleocapsid protein of peste des petits ruminants virus.

The nucleocapsid (N) protein of peste des petits ruminants virus (PPRV) with a conserved amino acid usage pattern plays an important role in viral replication. The primary objective of this study was to estimate roles of synonymous codon usages of PPRV N gene and tRNA abundances of host in the formation of secondary structure of N protein. The potential effects of synonymous codon usages of N gene and tRNA abundances of host on shaping different folding units (α-helix, ß-strand and the coil) in N protein were estimated, based on the information about the modeling secondary structure of PPRV N protein. The synonymous codon usage bias was found in different folding units in PPRV N protein. To better understand the role of translation speed caused by variant tRNA abundances in shaping the specific folding unit in N protein, we modeled the changing trends of tRNA abundance at the transition boundaries from one folding unit to another folding unit (ß-strand â†’ coil, coil â†’ ß-strand, α-helix â†’ coil, coil â†’ α-helix). The obvious fluctuations of tRNA abundance were identified at the two transition boundaries (ß-strand â†’ coil and coil â†’ ß-strand) in PPRV N protein. Our findings suggested that viral synonymous codon usage bias and cellular tRNA abundance variation might have potential effects on the formation of secondary structure of PPRV N protein.

[Determination of trace amounts of mercury by spectrophotometric method with 2-hydroxy-5-sulfobenzenediazoaminoazobenzene].

The chromogenic reaction of 2-hydroxy-5-sulfobenzenediazoaminoazobenzene (HSDAA) with mercury was studied. In the presence of Triton X-100 and in a borax-buffer solution of pH 10.30, mercury(II) reacted with HSDAA to form a stable orange-red complex with molar radio of 1:2. The apparent molar absorptivity was 1.67 x 10(5) x L mol(-1) x cm(-1) with the maximum absorption wavelength of 519 nm. Beer's law was obeyed in the concentration range of 0-600 microg x L(-1) for Hg(II). The method was applied to the determination of trace amounts of mercury in water with satisfactory results.

[Spectrophotometric study of color reaction of chromogenic reagent 2-hydroxy-5-sulfo-benzenediazoaminoazobenzene(HSDAA) with thallium(III)].

The chromogenic reaction of the 2-hydroxy-5-sulfo-benzenediazoaminoazobenzene(HSDAA) with thallium was studied in this paper. In the presence of Triton X-100 and in ammonia medium of 0.72-0.99 mol.L-1, HSDAA reacts sensitively with thallium (III) to form a red complex with molar radio of 1:2. The apparent molar absorptivity is 1.04 x 10(5) L.mol-1.cm-1 at the maximum absorption wavelength of 516 nm. Beer's law is obeyed in the concentration range of 0-0.8 mg.L-1 Tl(III). Adding NaC4H4O6 and NaCN in the system can improve selectivity of the chromogenic agent greatly. The method has been applied to the direct determination of trace amounts of thallium in sample of synthetic water with satisfactory results.

Immunization trials with an avian chlamydial MOMP gene recombinant adenovirus.

Chicks were inoculated with a live vector vaccine of avian chlamydial MOMP gene recombinant adenovirus to evaluate efficacy, safety and viability of the vaccine. Five batches of the recombinant adenovirus vaccines, which were prepared using the 22nd generation avian chlamydial MOMP gene recombinant adenovirus cultured in HEK293 cells, were used to vaccinate 7 days-old chicks negative for chlamydial antibody. The recombinant adenovirus vaccine was shown to be both safe and effective in inducing specific immunity in vaccinated chicks.

Serological detection of bovine ephemeral fever virus using an indirect ELISA based on antigenic site G1 expressed in Pichia pastoris.

An indirect ELISA for the serological detection of bovine ephemeral fever virus (BEFV) infection in cattle is described in which a glycosylated protein of approximately 25 kDa (including the G1 antigenic site of the virus glycoprotein) expressed in Pichia pastoris GS115 was used as the coating antigen. The optimal concentration of coated antigen was 0.3 microg/well at a serum dilution of 1:40. The optimal positive threshold value of the assay was 1.88, as derived from receiver operating characteristic curve analysis. The test had 100% sensitivity and 96.7% specificity when compared with a micro-neutralisation test using 336 positive and 180 negative sera to BEFV, respectively. The inter-assay and intra-assay coefficients of variation for 15 sera were both <5.8% and there was no evidence of cross-reactivity between the tested coating antigen and antibodies to related rabies virus. The ELISA is an inexpensive and rapid serological detection method that would be suitable for screening for BEFV infection on a large scale.

An indirect ELISA of classical swine fever virus based on quadruple antigenic epitope peptide expressed in E.coli.

In this study, a synthesized quadruple antigenic epitope gene region of the classical swine fever virus (CSFV) E2 glycoprotein was expressed in E. coli to a obtain target protein. This target protein was used as a coating antigen to establish an indirect ELISA for specifically detecting anti-CSFV antibodies in serum samples from pigs. The P/N cut-off value of this assay was 1.92 by receiver operating characteristic curve (ROC) analysis based on 30 negative sera and 80 positive samples. The test gave 97.5% sensitivity and 96.7% specificity compared with the indirect hemagglutination (IHA) test. The inter-assay and intra-assay coefficients of variation (CVs) for 16 sera were both ≤6.8%. No cross-reactivity between the coating antigen and anti-bovine viral diarrhoea virus (BVDV) antibodies was observed.

Development and application of G1-ELISA for detection of antibodies against bovine ephemeral fever virus.

The gene encoding antigenic site G(1) of bovine ephemeral fever virus (BEFV) was highly expressed in the host cell Escherichia coli. An indirect G(1)-ELISA with the recombinant protein as the coating antigen was established to detect antibodies against BEFV. The result revealed that the optimal concentration of the coated antigen was 0.5 microg/well and the dilution of serum was 1:20. It was optimal that sera with P/N >or= 2.2 were considered positive, P/N

Construction and immunogenicity of recombinant adenovirus expressing the major outer membrane protein (MOMP) of Chlamydophila psittaci in chicks.

Avian chlamydiosis is caused by Chlamydophila psittaci. The major outer membrane protein (MOMP) encoded by the outer membrane protein 1 (omp1) gene is an excellent candidate for genetic engineering of a vaccine against avian chlamydiosis. In this study, the MOMP gene was amplified by PCR and cloned into the transfer vector pShuttle-CMV. The recombinant plasmid was obtained by recombination between the plasmid pShuttle-CMV-MOMP and skeleton vector pAdEasy-1 in Escherichia coli strain BJ5183. The titer of recombinant adenovirus containing the MOMP gene (rAd-MOMP) of C. psittaci was 3.4x10(10)TCID(50)/ml in human embryonic kidney 293 (HEK293) monolayer cells. The expression of the MOMP in HEK293 cells infected with rAd-MOMP was confirmed by an indirect immunofluorescence assay. Specific pathogen free (SPF) chicks were inoculated with 10(6), 10(8), and 10(10)TCID(50) of rAd-MOMP/chick. Inoculated chicks generated antibodies against MOMP of C. psittaci, which were detected by an indirect hemagglutination test (IHA). The vaccinated chicks were challenged with a virulent Chinese field isolate. Nine out of 10 chicks in the vaccinated group were protected, while birds in the wild-type adenovirus control group and the PBS control group all showed clinical signs after challenge. The results indicate that the recombinant adenovirus containing the MOMP gene of C. psittaci might be a candidate vaccine against avian chlamydiosis.