فهرست مطالب n .mosavari

Burkholderia mallei is the main cause of glanders as a dangerous contagious zoonosis disease that is mostly observed in single-hoofed animals, especially horses. Modern molecular techniques have been recently employed to improve epidemiology for identifying and searching for strains of this bacterium at different times and locations. Due to the unknown number of circulating strains and lack of preventive methods, glanders is still observed in the form of epidemics. The present study aimed to evaluate six field isolates plus two laboratory strains of Borkolderia mallei and Burkholderia pseudomallei using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. All the isolates and strains were microbially cultured in the glycerol nutrient and glycerol agar media. The individually grown colonies of the bacterium were used in the biochemical tests. The DNA of isolates was extracted by boiling, and the PCR-RFLP test was conducted on their genome. Finally, the bacterium was injected into guinea pigs to induce the Straus reaction. The biochemical assays (or bioassays) confirmed the isolates as Burkholderia mallei. The PCR-RFLP assay demonstrated a product for Burkholderia mallei with a length of 650 bp. Nevertheless, 250 and 400 bp were produced for Burkholderia pseudomallei. The swollen scrotum pointed to the occurrence of the Straus reaction. The PCR-RFLP is a proper differential diagnosis technique for B. mallei; moreover, it is a suitable method for differentiating between Burkholderia mallei and Burkholderia pseudomallei. This technique can detect Burkholderia mallei in a short time with high precision and sensitivity.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis). The laboratory diagnosis of the disease includes various bacteriologic and immunologic methods. Despite the effectiveness of many of these methods in diagnosing active TB, their high cost and time-consuming nature have led researchers to adopt more accurate and rapid screening methods based on specific antigens for M. tuberculosis. The present study aimed to measure specific antibody serum levels against the early secretory antigenic target 6 kDa (ESAT-6) recombinant protein in healthy people and compare it to TB patients. The target population included 27 TB patients and 87 healthy individuals with no clinical TB symptoms. The healthy population was divided into two groups, including positive purified protein derivative (PPD) and negative PPD (35 and 52 people, respectively), using the Tuberculin skin test. The specific antibody level against the ESAT-6 recombinant antigen and the PPD protein was measured using an indirect Enzyme-Linked Immunosorbent Assay (ELISA) test. The results of the study showed that the majority of the healthy population with no symptoms of clinical TB and having negative skin test results did not have antibodies against the recombinant ESAT-6 (98%) and PPD (96%) antigens. On the other hand, there was a high level of the specific antibody of the ESAT-6 recombinant and PPD antigens in TB patients (77%). It is notable that in people with positive skin test results, the level of the antibody against the ESAT-6 recombinant antigen and PPD antigen was 94%. The results demonstrated that the ELISA method based on the measurement of antibodies against the ESAT-6 recombinant antigen can be a proper diagnostic method for rapid and accurate screening of healthy from infected people.

Recent advancements in drug delivery system through development of nanomedicine has significantly improved the delivery of the medicine to the target tissue, consequently reduced the required therapeutic dose, toxicity and side effects. The aim of this study was test antifungal susceptibility of trimethylchitosan nanoparticles in combination with amphotericin B (NPs-AmB) through broth micro-dilution method described in CLSA-A3 guidelines on resistant (R) and susceptible (S) Candida albicans isolates and the cell viability and proliferation assays. Minimum inhibitory concentrations (MICs) 90 % of NPs-AmB and AmB determined to be 0.25 and 0.75 µg/mL, and minimum fungicidal concentration (MFC) were 0.5 and 1 µg/mL for S-isolate. MIC 90 for R-isolate were 8 and 32 µg/mL and MFC were 32 and 64 µg/mL respectively demonstrating a significant improvement in antifungal activity (p

The presence of common zoonosis diseases caused by Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM), such as Johne's and Crohn's diseases, poses a public health threat and economic losses to Iranian livestock. Therefore, the early detection of mycobacteria is of paramount importance. In this regard, enzyme-linked immunosorbent assay (ELISA) is a new, simple to use, rapid, and useful diagnostic tool. This study was performed to evaluate different crude antigens obtained from Mycobacterium species using an indirect ELISA test to identify the mycobacterial infection in infected livestock. Five different strains of Mycobacteria including M. tuberculosis, M. phlei, M. bovis, M. avium subspecies paratuberculosis, and M. bovis AN5 were cultured. The crude antigens in the samples were precipitated with trichloroacetic acid 4%. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude antigens isolated from different Mycobacterium species was reported. The total level of protein was determined by the Lowry protein assay. After the crude antigen preparation, the ELISA test was performed and the results were compared with the purified protein derivative skin test. Data analysis was performed using SPSS software version 25. All five strains were detected in more than 92% of healthy animals. The highest sensitivity of ELISA tests was in M. bovis AN5 antigen which was greater than 83%. The highest diagnostic specificity and efficiency of assays were in M. avium subspecies paratuberculosis which was 95.83% and over 83%, respectively. Regarding the results, M. avium subspecies paratuberculosis and M. bovis AN5 antigens were promising candidates for the design of diagnostic ELISA due to their sensitivity, specificity, and efficiency.

Amphotericin B (AmB) is an effective antifungal agent; however, the application of AmB is associated with a number of drawbacks. Application of nanoparticles (NPs) is known to improve the efficiency of drug delivery to the target tissues, compared to the traditional methods. In this study, a novel method of NPs preparation was developed. The trimethyl chitosan (TMC) was synthesized using low molecular weight chitosan and was used for the preparation of TMC-NPs through ionic gelation method. Afterward, AmB-loaded TMC-NPs (TMC-NPs/AmB) were prepared and their drug delivery potential was testes. The TMC-NPs and TMC-NPs/AmB were characterized for their structure, particle size, Zeta potential, polydispersity index, morphology, loading efficiency, loading capacity, in vitro release profile, release kinetic, and entrapped AmB potency. The cytotoxicity and antifungal activity of TMC-NPs/AmB against Candida albicans biofilm were evaluated. The quaternization of TMC was estimated to be 36.4%. The mean particle size of TMC-NPs and TMC NPs/AmB were 210±15 and 365±10 nm, respectively, with a PDI of 0.30 and 0.4, ZP of +34±0.5 and +28±0.5 mV, respectively. Electron microscopy analysis indicated uniform spherical shapes with smooth surfaces. The TMC-NPs/AmB indicated LE of 76% and LC of 74.04 % with a potency of 110%. The release profile of TMC-NPs/AmB was best explained by the Higuchi model. The initial release after 10 h was obtained at 38%, and the rates of release after 36 and 84 h were determined at 67% and 76% respectively, which was significantly different (P<0.05) from previous time points. The minimum inhibitory concentration (MIC) (50%) of NPs/AmB and AmB were 0.65 and 1.75 μg/mL, and the MIC 80% were determined at 1.95 and 7.75 μg/mL, respectively, demonstrating a significant improvement in antifungal activity. The half-maximal inhibitory concentration for TMC-NPs/AmB and AmB were estimated at 86 and 105 μg/mL, respectively, indicating a significant reduction in cytotoxicity and the adverse effect. This study could successfully introduce a practical method to synthesize TMC-NPs. The encapsulation process was efficient and significantly improved the antifungal activity of AmB. The developed method can be applied to improve the feasibility of oral delivery while reducing the adverse effects associated with traditional methods.

In the last couple of years, a number of new and rapid tests for the diagnosis of Tuberculosis (TB) have been developed based on the low molecular weight antigens from Mycobacterium tuberculosis (Mtb) culture supernatant. This study aimed to isolate and purify low molecular weight antigens secreted by Mtb strain C for diagnostic purpose. The secretory proteins from culture filtrate of Mtb were extracted using ammonium sulphate precipitations and sephadex-G50 gel chromatography. The obtained antigen fractions were analyzed for their protein concentrations and approximate molecular weight using Lowry method and SDS-PAGE (12.5%), respectively. DOT-ELISA and Western blot assay was performed to confirm the presence of purified low molecular weight proteins isolated from Mtb using sera from pulmonary tuberculosis patients (polyclonal antibodies). During chromatography, low molecular weight proteins were separated, that was approximately 0.7 mg/ml of the total proteins (1.662 mg/ml). The purified protein fractions in molecular weight range of 14 kDa-41kDa appeared during SDS-PAGE analysis. The chromatographic band fraction in the weight range of 30-41 kDa was identified in the TB patients’ sera using Western blotting. The low molecular weight proteins in the culture filtrate of Mtb strain C were purified using ammonium sulphate and chromatography. These fractions were confirmed using Western blotting. The obtained results might support the hypothesis that the Mtb culture filtrate antigens could be used as a rapid and sensitive assay for the detection of patients with pulmonary TB.

Tuberculin skin test, also known as the tuberculin or purified protein derivative (PPD) test, is an extensively applied diagnostic test for the detection of primary infection with Mycobacterium tuberculosis (Mtb). The production of PPD is accompanied by some difficulties that require a series of modifications in the production and purification processes. The present study aimed to determine the facilitation level of the manufacturing process by modifying evaluation methods for the production of PPD tuberculin. Mtb strains were cultured in Lowenstein-Jensen media, and the cultured strains were inoculated into the Dorset-Henley liquid medium by the biphasic medium of potato-Dorset-Henley. After incubation, flasks containing cultured strain were selected for bacterial inactivation, and the optimal gamma radiation dose(s) was determined. Tuberculoproteins were precipitated by ammonium sulfate (AS) and Trichloroacetic acid (TCA). Protein concentration was determined using the Bradford and Kjeldahl protein assay methods. Finally, the lymphocyte transformation test and potency test were performed. Based on the results, the Dorset-Henley liquid medium is suitable for the massive growth of the bacterium. The transferal of Mtb from solid to liquid medium was directly carried out without intermediate culture. It was found that during tuberculoprotein production, heating at 100°C for 3 h would be safe for killing mycobacterium. Furthermore, the simultaneous use of heating and gamma irradiation (8 kGgy) killed all of the mycobacteria, while doses of 1, 1.5, and 7 kGy decreased a significant number of bacterial cells. The results also indicated that the concentration of tuberculoprotein extracted by TCA precipitation method was higher than that obtained by AS precipitation. The tuberculoproteins which were produced by these two methods in the lymphocyte transformation test were not significantly different in terms of potency (P>0.05). Moreover, due to the high volume of produced protein, the protein measurement was more efficiently carried out by the Kjeldahl method, compared to the Bradford method. Finally, the results of the present study demonstrated that in addition to the novel approach of gamma irradiation, optimum methods are efficient and applicable in the production of PPD tuberculin.

The members of the genus Mycobacterium, are the causative agents of mycobacteriosis, Mycobacteriosis is a chronic and progressive disease that may affect all tissues of the fish. Clinical signs include scale loss, dermal ulceration, spinal defects and etc. Mycobacteriosis is a zoonosis disease, and most people who deal with fish, are at risk. Due to the expansion of Rainbow fish breeding in the city of Sabzevar and having awareness the disease caused by Mycobacterium in fish and as a result of mortality and economic damage and also possibility of transferring this bacterium from fish to humans, it is necessary to study Mycobacterium in Sabzevar Rainbow fish. The purpose of this research is isolation and molecular identification of Mycobacterium from Rainbow fishes in Sabzevar pools. In this research 50 Rainbow fishes with clinical signs collected from seven different fish breeding pools in Sabzevar city. After autopsy and culture of the specimens in sterile conditions, in order to confirm acid-fast of the bacteria, was performed Ziel-Neelsen staining. Then, DNA extraction was performed and PCR-16s rRNA was performed to confirm mycobacterium infection. To determine identity, the results of the sequencing of the PCR product were evaluated using the BLAST program. In this research, three acidfast isolates grew, that the sequence of all three isolates belonged with 99% similar to Mycobacterium peregrinum. Given that M. peregrinum is isolated from aquatic and human respiratory tract infections, Therefore, it is necessary to warn people who deal with fish in any way.

Among several tuberculosis vaccine candidates for replacement of BCG، ESAT-6 protein has a special role. Since mycobacterium tuberculosis infection most often attacks the lungs، intranasal rout can be regarded as appropriate methods for tuberculosis vaccines and drug delivery. One of the appropriate systems for intranasal vaccine delivery is using biodegradable nanoparticles. Among biodegradable polymers، chitosan polymer has great features to increase the response of immunity system. This study aimed to investigate the specific humoral immune response of mice model after encapsulation of recombinant ESAT-6 antigen in chitosan nanoparticles.

The chitosan nanoparticles containing ESAT-6 antigen were synthesized by ionic gelation. Nanoparticle properties including morphology، particle size، zeta potential، encapsulation rates، and protein release were measured in vitro. The immunization was performed through the nose for 3 times on days 0 and 14 and 28. 2 weeks after last administration، blood samples were collected and specific IgG titers were measured by indirect ELISA.

The nanoparticles synthesized had appropriate properties. The mean size of resulting nanoparticles was 242. 8 nm by excellent antigen loading capacity (95. 23 %). The vitro release of antigen from nanoparticles after 200 hours was detected as 67. 5%. The Level of IgG antibody showed significant increase in the group that had received chitosan nanoparticles containing ESAT-6 compared with other groups.

ESAT-6 protein was encapsulated in chitosan nanoparticles successfully. Administration of chitosan nanoparticles can be a suitable method for administration of humoral immunity antigens of Mycobacterium tuberculosis through intranasal rout.