A specific reverse complement sequence for distinguishing Brucella canis from other Brucella species.

Canine brucellosis is primarily caused by Brucella canis, but other Brucella species can also cause the disease. Identifying sequences specific to B. canis and establishing PCR assays that can distinguish between B. canis and other Brucella species is essential to determine the etiology of canine brucellosis and the source of infection and to achieve effective control. We analyzed the gaps and SNPs of genomes I and II from B. canis strain RM6/66 and B. melitensis strain 16M using the Mauve genome alignment software, and the specificity of each of these differential regions was analyzed by BLAST. A 132 bp specific sequence was found between the DK60_915 (glycosyl hydrolase 108 family protein) and DK60_917 (aldose 1-epimerase) loci in B. canis chromosome 1. Further comparative analysis revealed that this is a reverse complement sequence between B. canis and other Brucella species. Then, three primers were designed based on the sequence that could detect B. canis with a 310 bp amplification product or other Brucella species with a 413 bp product. The PCR based on these primers had reasonable specificity and a sensitivity of 100 copies of Brucella DNA. The detection results for the blood samples of the aborted dogs showed a favorable accordance with the Bruce-ladder multiplex PCR assay. In conclusion, we found a specific reverse complement sequence between B. canis and other Brucella and developed a PCR method that allows a more comprehensive identification of the pathogen involved in canine brucellosis. These findings provide an effective means for preventing and controlling brucellosis.

Proteomic and Antibody Profiles Reveal Antigenic Composition and Signatures of Bacterial Ghost Vaccine of Brucella abortus A19.

Brucellosis is an important zoonotic disease that causes great economic losses. Vaccine immunisation is the main strategy for the prevention and control of brucellosis. Although live attenuated vaccines play important roles in the prevention of this disease, they also have several limitations, such as residual virulence and difficulty in the differentiation of immunisation and infection. We developed and evaluated a new bacterial ghost vaccine of Brucella abortus A19 by a new double inactivation method. The results showed that the bacterial ghost vaccine of Brucella represents a more safe and efficient vaccine for brucellosis. We further characterised the antigenic components and signatures of the vaccine candidate A19BG. Here, we utilised a mass spectrometry-based label-free relative quantitative proteomics approach to investigate the global proteomics changes in A19BGs compared to its parental A19. The proteomic analysis identified 2014 proteins, 1116 of which were differentially expressed compared with those in A19. The common immunological proteins of OMPs (Bcsp31, Omp25, Omp10, Omp19, Omp28, and Omp2a), HSPs (DnaK, GroS, and GroL), and SodC were enriched in the proteome of A19BG. By protein micro array-based antibody profiling, significant differences were observed between A19BG and A19 immune response, and a number of signature immunogenic proteins were identified. Two of these proteins, the BMEII0032 and BMEI0892 proteins were significantly different (P < 0.01) in distinguishing between A19 and A19BG immune sera and were identified as differential diagnostic antigens for the A19BG vaccine candidate. In conclusion, using comparative proteomics and antibody profiling, protein components and signature antigens were identified for the ghost vaccine candidate A19BG, which are valuable for further developing the vaccine and its monitoring assays.

Combined immunization with inactivated vaccine reduces the dose of live B. abortus A19 vaccine.

BACKGROUND: Brucella spp. is an important zoonotic pathogen responsible for brucellosis in humans and animals. Brucella abortus A19 strain is a widespread vaccine in China. However, it has a drawback of residual virulence in animals and humans. METHODS: In this study, the BALB/c mice were inoculated with either 100 µL PBS(control group, C group), 109 CFU/mL inactivated B. abortus A19 strain (I group), 105 CFU/mL (low-dose group, L group) 106 CFU/mL live B. abortus A19 strain (high-dose group, H group), or 105 CFU/mL live B. abortus A19 strain combined with 109 CFU/mL inactivated B. abortus A19 strain (LI group). Mice were challenged with B. abortus strain 2308 at 7 week post vaccination. Subsequently, the immune and protective efficacy of the vaccines were evaluated by measuring splenic bacterial burden, spleen weight, serum IgG, interferon-gamma (IFN-γ), interleukin-4 (IL-4) percentage of CD4 + and CD8 + T cells of mice via bacterial isolation, weighing, ELISA and flow cytometry, respectively. RESULTS: The splenic bacterial burden and spleen weight of the mice in group LI were mostly equivalent to the mice of group H. Moreover, Brucella-specific serum IgG, IFN-γ, IL-4, and the percentage of CD4+ and CD8+ T cells of the LI group mice were similar to those of the H group. In the subsequent challenge test, both vaccines conferred protective immunity to wild-type (WT) 2308 strain. In addition, the levels of IL-4 and IFN-γ, CD4+ and CD8+ T cells in these mice were similar to those of the mice in the H group. CONCLUSIONS: Combined immunization with low dose live vaccine and inactivated vaccine allowed to reduce the live B. abortus A19 vaccine, dose with an equivalent protection of the high-dose live vaccine.

Identification of severe fever with thrombocytopenia syndrome virus genotypes in patients and ticks in Liaoning Province, China.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS), caused by the SFTS virus (SFTSV), is an acute infectious disease transmitted by ticks that has recently been identified. There are no reports of epidemic serotypes in Liaoning Province, PR China. The aim of this study was, therefore, to identify genotypes of SFTSV in this province. METHODS: In 2019, quantitative PCR testing was performed on 17 patients suspected of being infected with SFTS in Liaoning Province and on 492 ticks from the counties and cities surrounding the patients' residences. Four samples were subjected to virus isolation and whole-genome amplification. RESULTS: Molecular diagnostic results confirmed SFTSV infection in five of the 17 suspected cases of SFTS and in 12 of the 492 ticks, with a prevalence of 2.4%. Four strains of SFTSV were successfully isolated from patients' blood and ticks. Phylogenetic analysis after whole-genome amplification and sequencing showed that they all belonged to genotype A of SFTSV. CONCLUSIONS: This study is the first to determine the genotype of SFTSV in patients and ticks in Liaoning Province, PR China. The results deepen our understanding of the SFTS epidemic and provide information on the variability in mortality rate among genotypes.

DNA Extraction with TRIzol Reagent Using a Silica Column.

TRIzol is a monophasic solution of phenol and guanidine isothiocyanate used for the extraction of RNA, DNA and proteins from tissues or cells. However, few studies have described its application to DNA extraction due to its time-consuming procedure. We present a TRIzol-modified method of extracting DNA from tissues using the TRIzol reagent and a silica column, which requires only one-third of the time required for the classic extraction procedure. Spectrophotometric analysis showed that the 260/280 and 260/230 nm optical density ratio of the DNA extracted using the TRIzol-modified method is ideal and equal to that obtained by the classic method and commercial DNAiso methods. The DNA extracted by the TRIzol-modified method had the same performance in a restriction enzyme digestion and quantitative PCR as that extracted using the classic method. Using the TRIzol-modified method saves time, simplifies the DNA extraction procedure, and facilitates various molecular biology assays.

High repetitive arginine in the anterior of PCV3 capsid protein is a severe obstacle for its expression in E. coli.

Porcine circovirus type 3 (PCV3) is a novel circovirus identified in sows with PDNS-like clinical signs and reproductive failure. The capsid protein (CAP) of PCV3 is expected to be an effective vaccine candidate. Here, we expressed the original capsid protein, truncated capsid protein without anterior highly repetitive arginine (ΔCAP) and their codon-optimized counterparts in E. coli. These results showed that lots of repeated arginine could severely lower the expression of PCV3 capsid protein in E. coli. At the same time, the recombined truncated PCV3 capsid protein forms typic virions. The efficient expression of capsid protein is expected to serve the development of PCV3 vaccines and other studies of PCV3 capsid protein.

Distribution of the COVID-19 epidemic and correlation with population emigration from Wuhan, China.

BACKGROUND: The ongoing new coronavirus pneumonia (Corona Virus Disease 2019, COVID-19) outbreak is spreading in China, but it has not yet reached its peak. Five million people emigrated from Wuhan before lockdown, potentially representing a source of virus infection. Determining case distribution and its correlation with population emigration from Wuhan in the early stage of the epidemic is of great importance for early warning and for the prevention of future outbreaks. METHODS: The official case report on the COVID-19 epidemic was collected as of January 30, 2020. Time and location information on COVID-19 cases was extracted and analyzed using ArcGIS and WinBUGS software. Data on population migration from Wuhan city and Hubei province were extracted from Baidu Qianxi, and their correlation with the number of cases was analyzed. RESULTS: The COVID-19 confirmed and death cases in Hubei province accounted for 59.91% (5806/9692) and 95.77% (204/213) of the total cases in China, respectively. Hot spot provinces included Sichuan and Yunnan, which are adjacent to Hubei. The time risk of Hubei province on the following day was 1.960 times that on the previous day. The number of cases in some cities was relatively low, but the time risk appeared to be continuously rising. The correlation coefficient between the provincial number of cases and emigration from Wuhan was up to 0.943. The lockdown of 17 cities in Hubei province and the implementation of nationwide control measures efficiently prevented an exponential growth in the number of cases. CONCLUSIONS: The population that emigrated from Wuhan was the main infection source in other cities and provinces. Some cities with a low number of cases showed a rapid increase in case load. Owing to the upcoming Spring Festival return wave, understanding the risk trends in different regions is crucial to ensure preparedness at both the individual and organization levels and to prevent new outbreaks.

Rapid and simple detection of Glaesserella parasuis in synovial fluid by recombinase polymerase amplification and lateral flow strip.

BACKGROUND: Glaesserella parasuis (G. parasuis) is an influential pathogen of the pig, which induces high morbidity and mortality in naive pig populations in the pig industry. Accurate and rapid detection of the agent is important for disease control. In this study, a simple recombinase polymerase amplification (RPA) with a Lateral flow (LF) strip (RPA-LF-GPS) was developed to detect G. parasuis. RESULTS: The RPA-LF-GPS can specifically detect G. parasuis a limit of 100 CFU from other common related pathogens causing arthritis in the pig. The RPA-LF-GPS assay can use boiled synovial fluid samples as a template with the same sensitivity as other DNA extraction methods. In the detection of clinic positive synovial fluid sample, RPA-LF-GPS is equally sensitive (98.1%) compared with that of PCR (90.4%) (P > 0.05). The whole procedure of the RPA-LF-GPS assay could be finished in 1 hour without professional equipment. CONCLUSIONS: RPA-LF-GPS assay is a rapid and simple method for point-of-care diagnostic testing for G. parasuis infection.

Treatment of Uncomplicated Recurrent Urinary Tract Infection with Chinese Medicine Formula: A Randomized Controlled Trial.

OBJECTIVE: To evaluate Chinese medicine (CM) formula Bazheng Powder () as an alternative therapeutic option for female patients with recurrent urinary tract infection (RUTI). METHODS: A randomized double-blinded trial was performed. Eligible female patients with RUTI were recruited from one hospital and two community health centers. By using a blocked randomization scheme, participants were randomized to receive a CM formula (10 herbs) for 4 weeks or antibiotics for 1 week, followed by 3 weeks of placebo. Clinical cure rate and microbiological cure and recurrence after treatment were evaluated. RESULTS: A total 122 eligible patients were enrolled, with 61 cases in each group. The clinical cure rate by the intentto- treatment approach was 90.2% for the CM group and 82.0% for the antibiotics group (P>0.05). Bacteria were cleared from 88.5% (54/61) of patients in the CM group and 82.0% (50/61) in the antibiotics group. The recurrence rate in recovered patients at the 6-month follow-up was 9.1% (5/61) and 14.0 (7/61) in the CM and antibiotics groups, respectively (P>0.05). CONCLUSION: CM formula Bazheng Powder is a good alternative option for RUTI treatment. (Registration No. NCT01745328).

Development and application of a simple recombinase polymerase amplification assay for rapid point-of-care detection of feline herpesvirus type 1.

Feline herpesvirus type 1 (FHV-1) is a highly contagious pathogen of domestic cats and other members of the family Felidae. Point-of-care diagnosis of persistent infection in cats is essential for control of its spread. A recombinase polymerase amplification (RPA) assay (RPA-LFD-FHV) combined with a lateral flow dipstrip (LFD) was developed that uses human body heat for incubation. Sensitivity was evaluated by testing a serial dilution of a control plasmid, and specificity was evaluated by testing related viruses. Swab samples from cats with suspected infection were tested by RPA-LFD-FHV, and the results were compared to those obtained by PCR to evaluate its clinical performance. The RPA-FLD-FHV assay was carried out successfully within 20 min, using body heat for incubation. The RPA-FLD-FHV had a detection limit of 103 copies of the FHV-1 gD gene, which was lower than that of PCR, which was 104 copies. The assay could detect templates of FHV-1 but not those of other feline and canine viruses. Viruses in boiled samples could be efficiently detected by the RPA-FLD-FHV. Thirty-one out of the 80 samples were positive by the RPA-FLD-FHV assay, whereas only 27 were positive by PCR. DNA sequencing confirmed that the four samples that were positive by RPA-FLD-FHV but negative by PCR were indeed positive. These results indicate that RPA-FLD-FHV is applicable for clinical use. The RPA-FLD-FHV assay is a simple, rapid, and reliable method for point-of-care diagnosis of FHV-1 infection.

Multi-drug resistant uropathogenic Escherichia coli and its treatment by Chinese medicine.

OBJECTIVES: To investigate the resistance and virulence profiles of uropathogenic Escherichia coli (UPEC) and its treatment by Chinese medicine (CM) Fuzheng Qingre Lishi Formula (, FQLF). METHODS: UPEC strains were isolated from recurrent urinary tract infections (UTIs) patients. Patient sensitivities to 17 antibiotics were tested by the disk diffusion method. Virulence genes were screened by plolymerase chain reaction. A mouse model was constructed using a multi-drug resistant and virulent UPEC strain and treated with FQLF or the antibiotic imipenem. The treatment efficacy was evaluated by bacterial clearance from urine and the urinary organs. RESULTS: A total of 90 UPEC strains were collected, and 94.4% of the isolates were resistant to at least 1 antibiotic. Approximately 66.7% of the UPEC strains were multi-drug resistant. More than one virulence gene was found in 85.6% of the isolates. The extended-spectrum ß-lactamases (ESBL)-positive strains were more resistant than the negative ones. The virulence gene number was positively correlated with the resistance number (P<0.05). A mouse model was successfully constructed using UPEC10. Treatment with either FQLF or antibiotics significantly cleared bacteria from the mouse urine after 14 days. In the untreated control, the bacteria lasted for 28 days. FQLF treatment of the UTI mouse model greatly reduced the bacterial number in the kidney and bladder, but could not completely clear the bacteria. CONCLUSIONS: Multi-drug resistance is common among UPEC isolates, and the resistance is positively related with virulence. FQLF could treat UPEC UTIs, but could not completely clear the bacteria from the host.

[Optimization of the conditions for protein chip preparation].

OBJECTIVE: To define the optimal conditions for preparing protein chips on aldehyde-coated slides. METHODS: The proteins were diluted in PBS containing 40% glycerol and spotted on aldehyde-coated slides. After the spots were dried for 1 hour at room temperature, the slides were stored at 4 degrees Celsius;. Following block and rinse, the slides were used for immunoassay and the results detected with a scanner. Several key factors that might influence the results were tested, including the number of spots, concentration of protein in the spotting solution, time of immobilization and the blocking reagent. RESULTS: Pre-spotting of 10 to 15 spots achieved good homogeneity of the subsequent spots on aldehyde-coated slides. The protein immobilized at 4 degrees Celsius; for 24 to 48 h showed higher fluorescence intensity and clearer images, and the slides blocked with 3% BSA produced the lowest background signal. The concentration of protein in the spotting solution significantly affected the fluorescence intensity. CONCLUSION: To ensure good results in preparing protein chips on aldehyde slides, pre-spotting of 10-15 spots can be necessary followed by immobilization at 4 degrees Celsius; for 24-48 h and 3% BSA blocking.