cRapid and effective diagnosis of brucellosis is a challenge for clinicians. Even when diagnosis is on time and therapy is initiated, meticulous follow-up appointments are crucial for ensuring the efficacy of the treatment. Due to shortcomings of serological methods, molecular diagnosis, especially real-time PCR, is becoming a main approach in laboratory diagnostics. Thus, the development of efficient procedures and standardization of the PCR tests will have a great impact on the precise detection and quantification of bacterial DNA loads, which is valuable for the medical management of brucellosis patients. We developed a new TaqMan real-time PCR directed to bcsp31, a shared gene of the brucellae. The bcsp31 gene fragment was cloned into pJET1.2. Recombinant pJET1.2-bcsp31 was linearized by HindIII digestion, and the product was used for the preparation of a standard curve. A panel of Brucella spp. and non-Brucella pathogens was tested. No bacterial genomes other than those of the brucellae were detected. According to the results, specificity of the method was 100%. In a clinical assessment, the positive-control group comprised 37 patients with microbiologically confirmed brucellosis, and 25 healthy individuals served as the negative-control group. By the end of the treatment period, there was a significant decrease in the DNA load of the 37 brucellosis patients, which persisted for the 4 weeks of monitoring after treatment, suggesting that our proposed method is an efficient monitoring tool. Serum samples prior to any treatment were collected from the 25 serologically suspicious patients and assessed by our method; 72% of these patients tested positive for brucellosis.T he brucellae are facultative, intracellular, Gram-negative bacteria that infect animals and humans (1). Although it has been eradicated in some developed countries, brucellosis still remains a major problem worldwide. Diagnosis of the disease presents a major challenge to clinical and veterinary services (2, 3). Brucella spp. are able to survive within host cells, leading to relapses and focal complications (1). Early diagnosis of brucellosis results in early initiation of therapy, which plays a key role in the success of the control and eradication programs; nonetheless, accurate diagnosis requires reliable and sensitive diagnostic tools (4). The intracellular localization of the bacteria and the slow evolution of the disease hamper the usefulness of blood cultures (5). Despite being the gold standard, isolation of brucellae from blood culture or other sterile body fluids has a sensitivity of about 70% (4, 6). Serodiagnostic assays are principally based on antibodies against the Brucella lipopolysaccharide (LPS) and have high sensitivity but low specificity. The low specificity may be due to the crossreaction of antibodies with LPS from other bacterial species and subsensitive or high-immunity reactions, depending on the subclinical or endemic prevalence of the disease (4, 6, 7). The presence of IgG and IgM for months after therapy complicates...
Control of brucellosis as a worldwide zoonotic disease is based on vaccination of animals and diagnosis of infected cases to be eradicated. Accurate and rapid detection of infected animals is of critical importance for preventing the spread of disease. Current detection of brucellosis is based on whole-cell antigens and investigating serum antibodies against Brucella lipopolysaccharide (LPS). The critical disadvantage is misdiagnosis of vaccinated animals as infected ones and also cross-reactions with other Gramnegative bacteria. Recombinant outer membrane protein 2b (Omp2b) of Brucella abortus was evaluated as a novel serodiagnostic target in comparison to conventional tests which are based on LPS. Recombinant Omp2b (rOmp2b) was expressed in Escherichia coli BL21 and purified by Ni 2+-based chromatography. rOmp2b was evaluated in an indirect enzyme-linked immunosorbent assay (ELISA) system for diagnosis of brucellosis, with sera from Brucella-infected mice along with negative sera and sera from mice which were inoculated with other Gram-negative species for assurance of specificity. Thereafter, cattle sera collected from different regions were assessed along with known negative and known positive serum samples. We found that Omp2b can discriminate between Brucella-infected animals and non-infected ones. Results for assessment of two hundred and fifty cattle sera by Omp2b-based indirect ELISA which were compared to Rose Bengal plate agglutination test (RBPT) and serum tube agglutination test (SAT) showed that our proposed procedure has the sensitivity of 88.5%, specificity of 100%, and accuracy of 90.8%. We suggest that recombinant Omp2b could be used as a protein antigen for diagnosis of brucellosis in domestic animals and can be evaluated for detection of human brucellosis.
Protein antigens have drawn a lot of attention from investigators working on tuberculosis vaccines. These proteins can be used to improve the immunogenicity of the new generation BCG vaccines or even replace them completely. Recombinant technology is used to insure the production of pure mycobacterial antigens in high quantities. Mycolyl transferase 85B (Ag85B) is a potent, mycobacterial antigen that significantly stimulates immune responses. Since Ag85B is an apolar protein, production of the water-soluble antigen is of interest. In this work, we report a systematic optimization strategy concerning cloning systems and purification methods, aiming at increasing the yield of recombinant Ag85B. Our optimized method resulted in a yield of 8 mg of recombinant Ag85B from 1 liter of induced culture (400 μg/ml) by using pET32a(+), Escherichia coli Rosseta-gami™(DE3) pLysS and a Ni-NTA agarose-based procedure and on-column re-solubilization. The purified recombinant Ag85B showed strong immunostimulating properties by inducing high levels of TNF-α, IFN-γ, IL-12, and IgG2a in immunized mice, therefore it can effectively be applied in TB vaccine researches.
PurposeFimH (the adhesion fragment of type 1 fimbriae) is implicated in uropathogenic Escherichia coli (UPEC) attachment to epithelial cells through interaction with mannose. Recently, some studies have found that UPEC can thrive intracellularly causing recurrent urinary tract infection (UTI). Almost all vaccines have been designed to induce antibodies against UPEC. Yet, the humoral immune response is not potent enough to overcome neither the primary UTI nor recurrent infections. However, DNA vaccines offer the possibility of inducing cell mediated immune responses and may be a promising preventive tool.Materials and MethodsIn this study, we employed two different open reading frames within mammalian (mam) and wild type (wt) codons of fimH gene. Optimized fragments were cloned in pVAX-1. Expression of the protein in COS-7 was confirmed by western blot analysis after assessing pVAX/fimH(mam) and pVAX/fimH(wt). The constructs were injected to BALB/c mice at plantar surface of feet followed by electroporation.ResultsThe mice immunized with both constructs following booster injection with recombinant FimH showed increased interferon-γ and interleukin-12 responses significantly higher than non-immunized ones (p<0.05). The immunized mice were challenged with UPEC and then the number of bacteria recovered from the immunized mice was compared with the non-immunized ones. Decreased colony count in immunized mice along with cytokine responses confirmed the promising immune response by the DNA vaccines developed in this study.ConclusionIn conclusion, DNA vaccines of UPEC proteins may confer some levels of protection which can be improved by multiple constructs or boosters.
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