Biosynthesis and Immunological Evaluation of a Dual-Antigen Nanoconjugate Vaccine Against Brucella melitensis

Brucellosis,caused by Brucella,is one of the most common zoonosis.However,there is still no vaccine for human use.Although some live attenuated vaccines have been approved for animals,the protection effect is not ideal.In this study,we developed a dual-antigen nanoconjugate vaccine containing both polysaccharide and protein antigens against Brucella.First,the antigenic polysaccharide was covalently coupled to the outer membrane protein Omp19 using protein glycan coupling technology,and then it was successfully loaded on a nano-carrier through the SpyTag/SpyCatcher system.After confirming the efficient immune activation and safety performance of the dual-antigen nanoconjugate vaccine,the potent serum antibody response against the two antigens and remarkable protective effect in non-lethal and lethal Brucella infection models were further demonstrated through different routes of administration.These results indicated that the dual-antigen nanoconjugate vaccine enhanced both T helper 1 cell(Th1)and Th2 immune responses and protected mice from Brucella infection.Furthermore,we found that this protective effect was maintained for at least 18 weeks.To our knowledge,this is the first Brucella vaccine bearing diverse antigens,including a protein and polysaccharide,on a single nanoparticle.Thus,we also present an attractive technology for co-delivery of different types of antigens using a strategy applicable to other vaccines against infectious diseases.

Identification of Brucella melitensis and Molecular Cloning of Its BP26 Gene into p ET24a(+) Vector

Brucella spp. are pathogenic to humans and domestic animal. Nowadays,there is no effective vaccine and control strategy in China. So,it is necessary to research effective vaccines for prevention and treatment of this disease. In order to deal with these,we isolated and identified the type of Brucella in Darhan Muminggan Joint Banner and Siziwang Banner of Inner Mongolia. Totally 26 samples of sheep blood which were positive in serological test,one sample of spleen from aborted and one sample of secretion from birth canal were isolated,and the 16 S r DNA genes of positive samples were sequenced. Phylogenetic analysis proved that there were four isolates similar to B. melitensis. As an important diagnostic antigens of B. melitensis,the BP26 gene was amplified. The BP26 gene was cloned into vector p ET24a( +) and conducted sequence analysis. The BP26 gene was 900 bp,with an open reading frame of 753 bp. The homology of BP26 gene with the vaccine strain M5 was 100%,and that with S2 and A19 vaccine strains was 99. 99%. These finding supported the development of BP26-based specific serodiagnostic test and vaccine for B. melitensis in China.

In vitro assay for the anti-brucella activity of medicinal plants against tetracycline-resistant Brucella melitensis

Brucellosis, a zoonosis caused by four species of brucella, has a high morbidity. Brucella melitensis is the main causative agent of brucellosis in both human and small ruminants. As an alternative to conventional antibiotics, medicinal plants are valuable resources for new agents against antibiotic-resistant strains. The aim of this study was to investigate the usage of native plants for brucellosis treatment. For this purpose, the anti-brucella activities of ethanolic and methanolic extracts of Salvia sclarea, Oliveria decumbens, Ferulago angulata, Vitex pseudo-negundo, Teucrium polium, Plantago ovata, Cordia myxa, and Crocus sativus were assessed. The activity against a resistant Br. melitensis strain was determined by disc diffusion method at various concentrations from 50-400 mg/ml. Antibiotic discs were also used as a control. Among the evaluated herbs, six plant (Salvia sclarea, Oliveria decumbens, Ferulago angulata, Vitex pseudo-negundo, Teucrium polium, and Crocus sativus) showed anti-brucella activity. Oliveria de- cumbens was chosen as the most effective plant for further studies. A tested isolate exhibited resistance to tetracycline, nafcillin, oxacillin, methicillin, and colistin. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values for Oliveria decumbens against resistant Br. melitensis were the same (5 mg/ml), and for gentamicin they were both 2 mg/ml. Time-kill kinetics for a methanolic extract of Oliveria decumbens was 7 h whereas for an ethanolic extract it was 28 h. Also, Oliveria decumbens extracts showed a synergistic effect in combination with doxycycline and tetracycline. In general, the similar values of MIC and MBC for Oliveria decumbens suggest that these extracts could act as bactericidal agents against Br. melitensis. In addition to Oliveria decumbens, Crocus sativus and Salvia sclarea also had good anti-brucella activity and these should be considered for further study.

Epidemiologically characteristics of human brucellosis and antimicrobial susceptibility pattern of Brucella melitensis in Hinggan League of the Inner Mongolia Autonomous Region,China

Background:Hinggan League is located in the Northeast of the Inner Mongolia Autonomous Region,the historically endemic area of animal and human brucellosis.In this study,the epidemiological characteristics of human brucellosis were analyzed,and the genotypic profile and antimicrobial susceptibilities of Brucella melitensis strains isolated from humans in Hinggan League were investigated.Methods:The epidemic characteristics were described using case number,constituent ratio,and rate.The 418 human blood samples were collected and tested by bacteriology,and suspect colonies were isolated and identified by conventional biotyping assays,the VITEK 2.0 microbial identification system,and AMOS(Brucella abortus,B.melitensis,B.ovis,and B.suis)-PCR.Subsequently,all strains were genotyped using multiple-locus variable-number tandem repeat analysis(MLVA)assays,and the antimicrobial susceptibility pattern of Brucella strains against the 10 most commonly used antibiotics was determined by microdilution method.Results:A total of 22848 cases of human brucellosis were reported from 2004 to 2019,with an annual average incidence of 87.2/100000.The incidence rates in developed areas of animal husbandry(Horqin Youyi Qianqi[161.2/100000]and Horqin Youyi Zhongqi[112.1/100000])were significantly higher than those in forest areas(Arxan[19.2/100000])(χ2=32.561,P<0.001).In addition,peak morbidity occurred during May–August,accounting for 72.6%(16582/22848)of cases.The highest number of cases occurred in the 40+age group,accounting for 44.4%(10137/22484)of cases,and morbidity in males was significantly higher than that in females in all age groups(χ2=299.97,P<0.001),the most common occupation was farmers.A total of 54 B.melitensis strains were divided into 37 genotypes(GT1–37)with 80–100%genetic similarity.All 25 strains were sensitive to seven tested antibiotics,phenotypic resistance to cotrimoxazole and azithromycin was observed in 5(20%)and 25(100%)of the isolates,respectively.Conclusions:Human brucellosis exhibited a significant increasing trend and B.melitensis is the main pathogen responsible for human brucellosis in this region.Improved surveillance of infected animals(sheep)and limiting their transfer and trade are optional strategies for decreasing the incidence of this disease.

A case report of spontaneous abortion caused by Brucella melitensis biovar 3

Background:Brucellosis is a worldwide zoonotic disease caused by Brucella spp.Brucella invades the body through the skin mucosa,digestive tract,and respiratory tract.However,only a few studies on human spontaneous abortion attributable to Brucella have been reported.In this work,the patient living in Shanxi Province in China who had suffered a spontaneous abortion was underwent pathogen detection and Brucella melitensis biovar 3 was identified.Case presentation:The patient in this study was 22 years old.On July 16,2015,she was admitted to Shanxi Grand Hospital,Shanxi Province,China because of one day of vaginal bleeding and three days of abdominal distension accompanied by fever after five months of amenorrhea.A serum tube agglutination test for brucellosis and blood culture were positive.At the time of discharge,she was prescribed oral doxycycline(100 mg/dose,twice a day)and rifampicin(600 mg/dose,once daily)for 6 weeks as recommended by the World Health Organization(WHO).No recurrence was observed during the six months of follow-up after the cessation of antibiotic treatment.Conclusions:This is the first reported case of miscarriage resulting from Brucella melitensis biovar 3 isolated from a pregnant woman who was infected through unpasteurized milk in China.Brucellosis infection was overlooked in the Maternity Hospital because of physician unawareness.Early recognition and prompt treatment of brucellosis infection are crucial for a successful outcome in pregnancy.

RNA-seq reveals the critical role of Csp A in regulating Brucella melitensis metabolism and virulence

Brucella melitensis is a facultative intracellular bacterium that replicates within macrophages. The ability of Brucella to survive and multiply in the hostile environment of host macrophages is essential for its virulence. The cold shock protein Csp A plays an important role in the virulence of B. melitensis. To analyze the genes regulated by Csp A, the whole transcriptomes of B. melitensis NI?csp A and its parental wild-type strain, B. melitensis NI, were sequenced and analyzed using the Solexa/Illumina sequencing platform. A total of 446 differentially expressed genes were identified, including 324 up-regulated and 122 down-regulated genes. Numerous genes identified are involved in amino acid, fatty acid, nitrogen, and energy metabolism. Interestingly, all genes involved in the type IV secretion system and Lux R-type regulatory protein Vjb R were significantly down-regulated in NI?csp A. In addition, an effector translocation assay confirmed that the function of T4 SS in NI?csp A is influenced by deletion of the csp A gene. These results revealed the differential phenomena associated with virulence and metabolism in NI?csp A and NI, providing important information for understanding detailed Csp A-regulated interaction networks and Brucella pathogenesis.

A retrospective survey of Brucella melitensis human infection in Hainan Province,China

In China,brucellosis has spread from the northern provinces to the southern coastal area,and Brucella melitensis has become the overwhelmingly dominant species nationwide.Although human Brucellosis cases have been reported in many parts of Hainan Province,investigations into the source of infection have been rare.In this case,we have used the techniques of bacteriology,genome sequencing,and WGS-SNP to investigate the source of infection on a patient with a traveling history between Hainan and Inner Mongolia autonomous regions.We collected a 5mL blood sample under sterile conditions from this suspected case of Brucellosis infection and incubated it in a culture bottle.A week later,Brucella colonies were purified and identified under the standard bacteriology procedures to show that the strain appeared as B.melitensis bv.3.Subsequently,a draft genome sequencing of this strain was obtained.AWGS-SNP comparison analysis of Chinese B.melitensis selected(n=36)fromGenBank revealed that the strain in this studywas more similar to the strains isolated from China's northern parts,especially those from the Inner Mongolia Autonomous Region.These data indicated that the source of infection of the patient was in the Inner Mongolia Autonomous Region,but the actual location where this patient became infected was unclear.We suggest that travelers from the southern part of China be cautious when visiting the traditional endemic areas of Brucellosis.

Species and Biotype Analysis of Brucella Strains Isolated from Cattle and Sheep in Xinjiang

[ Objective] The paper aimed to understand the epidemiological characteristics of Brucella strains in Xinjiang and then provide an available integrated measure to prevent and control brucellosis. [ Method ] Eleven suspected Brucella strains were isolated by traditional methods, which were further identified by AMOS-PCR assay. Conventional biochemical tests were carried out to identify the biological subtype of sheep Brucella. [ Result] Nine strains were all B. meliten- s/s, and biological test indicated that all of them were B. melitensis biotype 3. [ Conclusion] B. melitensis biotype 3 was the predominant strain of Brucella in Xin- jiang, and AMOS-PCR assay could be applied safely and quickly as an assistant tool to detect Brucella. The results of molecular epidemiology laid a foundation for updating prevention and control strategy against brucellosis in Xinjiang.

Unusual causes of intrahepatic cholestatic liver disease

We report five cases with unusual causes of intrahepatic cholestasis, including consumption of Teucrium polium （family Lamiaceae） in the form of tea, Stauffer＇s syndrome, treatment with tamoxifen citrate for breast cancer, infection with Coxiella Burnetii （acute Q fever）, and infection with Brucella rnelitensis （acute brucellosis）.