Genome-wide transcription start site mapping in the facultative intracellular pathogen Brucella melitensis by Capping-seq.

Brucella melitensis (B. melitensis) is an important facultative intracellular bacterium that causes global zoonotic diseases. Continuous intracellular survival and replication are the main obstruction responsible for the accessibility of prevention and treatment of brucellosis. Bacteria respond to complex environment by regulating gene expression. Many regulatory factors function at loci where RNA polymerase initiates messenger RNA synthesis. However, limited gene annotation is a current obstacle for the research on expression regulation in bacteria. To improve annotation and explore potential functional sites, we proposed a novel genome-wide method called Capping-seq for transcription start site (TSS) mapping in B. melitensis. This technique combines capture of capped primary transcripts with Single Molecule Real-Time (SMRT) sequencing technology. We identified 2,369 TSSs at single nucleotide resolution by Capping-seq. TSSs analysis of Brucella transcripts showed a preference of purine on the TSS positions. Our results revealed that -35 and -10 elements of promoter contained consensus sequences of TTGNNN and TATNNN, respectively. The 5' ends analysis showed that 57% genes are associated with more than one TSS and 47% genes contain long leader regions, suggested potential complex regulation at the 5' ends of genes in B. melitensis. Moreover, we identified 52 leaderless genes that are mainly involved in the metabolic processes. Overall, Capping-seq technology provides a unique solution for TSS determination in prokaryotes. Our findings develop a systematic insight into the primary transcriptome characterization of B. melitensis. This study represents a critical basis for investigating gene regulation and pathogenesis of Brucella.

Microbiome analysis reveals the effects of black soldier fly oil on gut microbiota in pigeon.

The gut microbiota plays a vital roles in poultry physiology, immunity and metabolism. Black soldier fly oil is known to have a positive effect on the gut microbiota. However, the specific effect of black soldier fly oil on the composition and structure of the gut microbiota of the pigeon is unknown. In this experiment, 16S rDNA high-throughput sequencing was performed to study the effect of different doses of black soldier fly oil on the changes of pigeon intestinal microbes. Results indicated that the different doses of black soldier fly oil had no effect on the gut microbial diversity of the pigeon. Although the dominant phyla (Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria) and genus (uncultured_bacterium_f_Lachnospiraceae and Desulfovibrio) in control group and experimental group with different doses were the same, the abundances of some beneficial bacteria (Megasphaera, Intestinimonas, Prevotella_9, Lachnospiraceae_UCG-001, Faecalibacterium, Coprococcus_2, Parabacteroides, Megasphaera, Leuconostoc, Prevotellaceae_UCG-001, Lactococcus, Ruminococcaceae_UCG-014, and Coprococcus_2) increased significantly as the concentration of black soldier fly oil increased. Taken together, this study indicated that black soldier fly oil supplementation could improve gut microbial composition and structure by increasing the proportions of beneficial bacteria. Notably, this is the first report on the effects of black soldier fly oil on the gut microbiota of pigeon, which contribute to understanding the positive effects of black soldier fly oil from the gut microbial perspective.

[Prokaryotic expression and immunization of glutamyl tRNA synthetase of Streptococcus suis].

OBJECTIVE: Glutamyl tRNA Synthetase (Gts) is a protease which catalyzes esterification between tRNA and glutamine. The immunogenicity of Gts was evaluated through immunization and challenge experiment. METHODS AND RESULTS: We cloned gts from the genome of SC19, and inserted it into prokaryotic expression plasmid pET28a-gts. The recombinant vector was transformed into E. coli BL21. Induced by IPTG, one 58 kD protein, was expressed and purified by using Ni - NTA column (Novagen). The purity of rGtS was 93.3% and the concentration of purified protein was 433 microg/mL. We proved the immunogenicity of recombinant protein rGtS by western blot analysis. We immunized Balb/c mice with rGtS and Freund's adjuvant, and after two boost vaccinations, all mice were challenged with 4 times LD50 amount of SC19 (1.2 x 10(9) CFU). The survive rate of vaccination group is 50% (4/8), significantly higher than blank vector control group (1/8). CONCLUSION: These results proved that GtS has certain immunogenicity and can offer partial protection against high dose challenge. Therefore Gts could be a potential candidate of subunit vaccine against Streptococcus suis.

Comparison of the Intestinal Microbial Community in Ducks Reared Differently through High-Throughput Sequencing.

Birds are an important source of fecal contamination in environment. Many of diseases are spread through water contamination caused by poultry droppings. A study was conducted to compare the intestinal microbial structure of Shaoxing ducks with and without water. Thirty 1-day-old Shaoxing ducks (Qingke No. 3) were randomly divided into two groups; one group had free access to water (CC), while the other one was restricted from water (CT). After 8 months of breeding, caecal samples of 10 birds from each group were obtained on ice for high-throughput sequencing. A total of 1507978 valid sequences were examined and clustered into 1815 operational taxonomic units (OTUs). At phylum level, Firmicutes (41.37%), Bacteroidetes (33.26%), Proteobacteria (13.67%), and Actinobacteria (8.26%) were found to dominate the microbial community in CC birds, while Firmicutes (53.62%), Bacteroidetes (33.06%), and Actinobacteria (11.13%) were uncovered to be the prime phyla in CT ducks. At genus level, Bacteroides (25.02%), Escherichia-Shigella (11.02%), Peptococcus (7.73%) and Parabacteroides (5.86%) were revealed to be the mainly genera in the CC group ducks, while Bacteroides (18.11%), Erysipelatoclostridium (10.94%), Ruminococcaceae_unclassified (10.43%), Lachnospiraceae_unclassified (5.26%), Coriobacteriales_unclassified (5.89%), and Faecalibacterium (4.2%) were detected to staple the microbial flora in the CT birds. One phylum and 13 genera were found to have the significant difference between the two bird groups (p<0.05). At phylum level, Proteobacteria in CT ducks were found to be obviously lower than ducks in CC birds (p<0.05). At genus level, Escherichia-Shigella (p<0.05) and Peptococcus (p<0.05) were found to be notably lower in CT birds, while Erysipelatoclostridium (p<0.05), Ruminococcaceae_unclassified (p<0.01), Coriobacteriales_unclassified (p<0.05), Faecalibacterium (p<0.01), Atopobiaceae_unclassified (p<0.01), Alistipes (p<0.05), Eggerthellaceae_unclassified (p<0.05), Prevotella_7 (<0.05), Rikenellaceae_RC9_gut_group (p<0.05), Prevotellaceae_uncultured (p<0.05), and Shuttleworthia (p<0.05) were observed to be prominently higher in CT ducks. In conclusion, the present study revealed the effects of keeping ducks away from swimming with obvious changes in the microbial community. Though higher microbial richness was found in the ducks without swimming, more pathogenic genera including Eggerthella, Erysipelatoclostridium, Alistipes, Prevotella_7, and Shuttleworthia; zoonotic genera including Eggerthella and Shuttleworthia; inflammatory genus Alistipes; anti-inflammatory Faecalibacterium genus; and tumor genus Rikenellaceae were examined in these ducks. The CT ducks also showed significant changes at genera level regarding the metabolism (Peptococcus, Ruminococcaceae, and Coriobacteriales).

[Expression of prn gene of Bordetella bronchiseptica and development of a recombinant protein-based indirect ELISA for antibodies detection].

OBJECTIVE: We developed an indirect ELISA method for detecting Bordetella bronchiseptica (Bb) pertactin antibodies based on the recombinant pertactin protein expressed in Escherichia coli (DE3) strain. METHODS AND RESULTS: The prn gene encoding Bb pertactin was fused to the downstream of glutathione S-transferase (GST) of pGEX-KG expression vector, resulting in the fusion expression plasmid pGEX-prn. SDS-PAGE showed that the GST-PRN fusion protein was expressed in high level in BL21 carrying pGEX-prn. The strong reactivity of the GST-PRN fusion protein, specifically with antiserum against porcine Bordetellosis caused by Bb HH0809, was identified by Western blot. The recombinant protein fragment of rPRN was purified from the GST-PRN fusion protein digested by protease thrombin with the purity of 93.1%. The rPRN-based indirect ELISA was developed for detecting antibodies against PRN. The ELISA could detect positive samples in experimentally infected pigs fourteen days post inoculation and the degree of sensitivity was over 4 times higher than the latex agglutination test with the coating antigen of killed Bb. Thirty-two point seven percent of positive samples were detected in 1,229 clinical samples while no false positive results were found in detecting 7 antisera against porcine bacterial diseases. Sera samples from two bordetellosis-positive pig fields were tested by the indirect ELISA method and the results indicated that pigs were infected by Bb during the nursery periods. CONCLUSION: The assay showed excellent specificity, sensitivity and reduplication, and can be useful for epidemiological survey and clinical diagnosis of swine bordetellosis.

[Protecting mice from fatal Bordetella bronchiseptica infection by immunization with recombinant pertactin antigens].

OBJECTIVE: We evaluated the efficacy of the recombinant pertactin (PRN)-specific active or passive immunization against Bordetella bronchiseptica (Bb) in an aerosol challenge model established by using BALB/c mice. METHODS AND RESULTS: Mice, immunized subcutaneously with two doses of purified glutathione S-transferase (GST)-PRN protein mixed with an equal volume of Freund's adjuvant, produced robust PRN-specific IgG antibody activity. All 20 mice vaccinated with GST-PRN protein survived aerosol challenge with three times the 50% lethal dose (LD50) of virulent Bb HH0809 compared with 3 of 20 GST protein-treated controls and 4 of 20 PBS-treated controls that survived. Furthermore, we observed complete protection against intraperitoneal challenge with ten times the LD50 of virulent HH0809 strain in mice that were injected intraperitoneally with 0.5 ml rabbit anti-GST-PRN serum. No survivors were observed in mice that received either rabbit anti-GST serum or PBS alone. CONCLUSION: The recombinant PRN protein had strong immunogenicity against Bb infection.