Single-walled carbon nanotube-protein complex: A strategy to improve the immune response to protein in mice.

Vaccines represent an effective tool for controlling disease infection. As a key component of vaccines, many types of adjuvants have been developed and used today. This study is designed to investigate the efficacy of single-walled carbon nanotubes (SWCNTs) as a new adjuvant. The results showed that SWCNT could adsorb the antigen by intermolecular action, and the adsorption rate was significantly higher after dispersion of the SWCNTs in a sonic bath. The titer of specific antibody of mice in the SWCNTs group was higher than that of the mice in the antigen control group, confirming the adjuvant efficacy of SWCNTs. During immunisation, the specific antibody was detected earlier in the mice of the SWCNTs group, especially when the amount of antigen was reduced. And it was proved that the titer of antibodies was higher after subcutaneous and intraperitoneal injection compared to intramuscular injection. Most importantly, the mice immunised with SWCNTs showed almost the same level of immunity as the mice in the FCA (Freund's complete adjuvant) group, indicating that the SWCNTs were an effective adjuvant. In addition, the mice in the SWCNT group maintained antibody levels for 90 days after the last booster vaccination and showed a good state of health during the observed period. We also found that the SWCNTs were able to induce macrophages activation and enhance antigen uptake by mouse peritoneal macrophages.

Isolation and molecular characteristics of a recombinant feline calicivirus from Qingdao, China.

Feline calicivirus (FCV) is a highly contagious pathogen seriously affecting the upper respiratory tract and producing oral diseases in the feline. Despite widespread vaccination, the prevalence of FCV remains high. In this study, the FCV qingdao (qd)/2019/china was isolated from a domestic feline oropharyngeal swab collected from Qingdao, China. The virus was purified using the plaque assay and identified using the Polymerase chain reaction and indirect immunofluorescence assay methods, the capsid amino acid, VP1 of qd/2019/china, showed sequence identity with the other isolates ranging between 83.90% (ym3/2001/jp) and 91.10% (CH-JL4). The sequence of the capsid amino acid revealed qd/2019/china to be closely related to CH-JL4 and clustered with CH-JL4 in the phylogenetic tree. The phylo-genetic analysis indicated that the complete genomes (GenBank® accession No. MZ322896) of qd/2019/china and CH-JL4 were also classified into the same cluster. The recombination analysis with Simplot indicated that the qd/2019/china originated from the recombination of CH-JL4 and HRB-SS, and the region 3,821 - 5,301 nt originated from HRB-SS. Further, the region 3,821 - 5,301 nt were found to belong to the protease-polymerase (PP) of HRB-SS. Here, we isolated a new recombinant virus, FCV qd/2019/china. Therefore, these results would be beneficial for better understanding of the evolution and epidemiology of FCV.

A novel approach for detection of brucella using a real-time recombinase polymerase amplification assay.

Brucella, the etiological agent of brucellosis, is an important zoonosis pathogen worldwide. Brucella infects humans and various domestic and wild animals, and represents a great threat to public health and animal husbandry. In the present study, we developed a real-time recombinase polymerase amplification (RPA) assay for the detection of Brucella. The assay targeted the bcsp31 gene of Brucella, and an RPA exo probe and a pair of primers were selected for assay validation. RPA sensitivity and specificity were evaluated using plasmid standards, Brucella representative strains, and non-Brucella strains. The RPA assay achieved a detection limit of 17 molecules in 95% of cases based on probit analysis, and could successfully distinguish 18 representative Brucella strains (B. abortus biovars 1, 2, 3, 4, 5, 6, 7 and 9, B. melitensis biovars 1, 2 and 3, B. suis biovars 1, 2, 3 and 4, B. canis, B. neotomae and B. ovis), and four Brucella vaccine strains (A19, S19, S2 and M5). A total of 52 Brucella field strains were detected by real-time PCR and RPA in parallel, and compared with real-time PCR, the sensitivity of the RPA assay was 94% (49/52). Thus, this RPA assay may be a rapid, sensitive, and specific tool for the prevention and control of Brucellosis.

A rapid minor groove binder PCR method for distinguishing the vaccine strain Brucella abortus 104M.

BACKGROUND: Brucellosis is a widespread zoonotic disease caused by Gram-negative Brucella bacteria. Immunisation with attenuated vaccine is an effective method of prevention, but it can interfere with diagnosis. Live, attenuated Brucella abortus strain 104M has been used for the prevention of human brucellosis in China since 1965. However, at present, no fast and reliable method exists that can distinguish this strain from field strains. Single nucleotide polymorphism (SNP)-based assays offer a new approach for such discrimination. SNP-based minor groove binder (MGB) and Cycleave assays have been used for rapid identification of four Brucella vaccine strains (B. abortus strains S19, A19 and RB51, and B. melitensis Rev1). The main objective of this study was to develop a PCR assay for rapid and specific detection of strain 104M. RESULTS: We developed a SNP-based MGB PCR assay that could successfully distinguish strain 104M from 18 representative strains of Brucella (B. abortus biovars 1, 2, 3, 4, 5, 6, 7 and 9, B. melitensis biovars 1, 2 and 3, B. suis biovars 1, 2, 3 and 4, B. canis, B. neotomae, and B. ovis), four Brucella vaccine strains (A19, S19, S2, M5), and 55 Brucella clinical field strains. The assay gave a negative reaction with four non-Brucella species (Escherichia coli, Pasteurella multocida, Streptococcus suis and Pseudomonas aeruginosa). The minimum sensitivity of the assay, evaluated using 10-fold dilutions of chromosomal DNA, was 220 fg for the 104M strain and 76 fg for the single non-104M Brucella strain tested (B. abortus A19). The assay was also reproducible (intra- and inter-assay coefficients of variation = 0.006-0.022 and 0.012-0.044, respectively). CONCLUSIONS: A SNP-based MGB PCR assay was developed that could straightforwardly and unambiguously distinguish B. abortus vaccine strain 104M from non-104M Brucella strains. Compared to the classical isolation and identification approaches of bacteriology, this real-time PCR assay has substantial advantages in terms of simplicity and speed, and also reduces potential exposure to live Brucella. The assay developed is therefore a simple, rapid, sensitive, and specific tool for brucellosis diagnosis and control.

Genetically modified rabies virus ERA strain is safe and induces long-lasting protective immune response in dogs after oral vaccination.

Oral immunization in free-roaming dogs is one of the most practical approaches to prevent rabies for developing countries. The safe, efficient and long-lasting protective oral rabies vaccine for dogs is highly sought. In this study, rabies virus (RABV) Evelyn-Rokitnicki-Abelseth (ERA) strain wild-type (rERA) and a genetically modified type (rERAG333E) containing a mutation from arginine to glutamic acid at residue 333 of glycoprotein (G333E) were generated by reverse genetic. The recombinant virus rERAG333E retained growth properties of similar to the parent strain rERA in BHK-21 cell culture. The G333E mutation showed genetic stability during passage into neuroblastoma cells and in the brains of suckling mice and was significantly reduced the virulence of rERA in mice. rERAG333E was immunogenic in dogs by intramuscular inoculation. Mice orally vaccinated with rERAG333E induced strong and one year longer virus neutralizing antibodies (VNA) to RABV, and were completely protected from challenge with lethal street virus at 12months after immunization. Dogs received oral vaccination with rERAG333E induced strong protective RABV VNA response, which lasted for over 3years, and moderate saliva RABV-specific IgA. Moreover, sizeable booster responses to RABV VNA were induced by a second oral dose 1year after the first dose. These results demonstrated that the genetically modified ERA vaccine strain has the potential to serve as a safe and efficient oral live vaccine against rabies in dogs.