IFN-gamma and IL-2 responses to recombinant AlaDH against ESAT-6/CFP-10 fusion antigens in the diagnosis of latent versus active tuberculosis infection

Background: Discriminating latent tuberculosis infection [LTBI] from active TBI may be challenging. The objective of this study was to produce the recombinant L-alanine dehydrogenase [AlaDH] antigen and evaluate individuals with LTBI, those with active TBI, and uninfected individuals by enzyme-linked immunospot assay [ELISPOT] in order to distinguish LTBI from active TBI

Methods: This exploratory study was performed in the Iranian city of Shiraz from 2014 to 2015. The study population [N=99] was divided into 3 groups: individuals with newly diagnosed active TBI [n=33], their household contacts [n=33], and controls [n=33]. AlaDH was produced through PCR and cloning methods. The diagnostic characteristics of AlaDH vs. ESAT-6/ CFP-10 were evaluated in responses to interferon-gamma [IFN-gamma] and interleukin-2 [IL-2] with ELISPOT. Differences between the groups were assessed with the Kruskal-Wallis and Mann- Whitney tests for nonparametric data analysis. The statistical analyses were performed with SPSS, version 16

Results: IFN-gamma responses to both ESAT-6/CFP-10 [P=0.81] and AlaDH [P=0.18] revealed that there were no significant differences between the individuals with LTBI and those with active TBI. The same results were determined for IL-2 responses to ESAT-6/CFP-10 between the 2 groups, while significantly higher IL-2 responses to AlaDH were observed in LTBI than in active TBI. According to the ROC curve analysis, a cutoff value of 275 SFC showed sensitivity of 75.8% and specificity of 78.8% for distinguishing LTBI from active TBI by IL-2 responses to AlaDH

Conclusion: The current study suggests that it may be possible to discriminate LTBI from active TBI by IL-2 responses to AlaDH

Molecular characterization of mycobacterium bovis isolates with high copy number of IS6110 by restriction fragment length polymorphism

Mycobacterium bovis is the causative agent of bovine tuberculosis and belongs to the Mycobacterium tuberculosis complex. M. bovis usually carries only one or a few copies of the insertion sequence IS6110 in its genome. The aim of this study was evaluation of copy number of IS6110 in M. bovis isolates by restriction fragment length polymorphism [RFLP] method. In this experimental study, 25 lymph node specimens of tuberculin-positive cattle were collected and cultured by standard methods, afterward genomic DNA was extracted by chloroform-isoamyl alcohol. Genetic studies were conducted by Pvull and DNA hybridization with IS6110. Two isolates displayed more than of 4 copies of IS6110 by RFLP [IS6110-RFLP] method. The results of this study are unique and specific in Iran but reported in the world rarely. Therefore the new strains of M. bovis imported to Iran from other countries of the world

Quantiation of IL-4, IL-10 and IFN-gamma genes expression after immunization of mice with CFP-10 and ESAT-6 containing vectors

Tuberculosis is a disease with high morbidity, caused mainly by Mycobaterium tuberculosis [M.tb.]. DNA vaccines show a promising future due to their unique advantages over conventional methods. The early-secreted antigen target [ESAT]-6 and culture filtrate protein [CFP]-10 of M.tb. antigens have been identified as vaccine candidates against Mycobacteria and used as subunit vaccines, DNA or protein, in different studies. To investigate the potential of pcDNA3.1+ plasmid containing CFP-10 and ESAT-6 genes in induction of local immune responses after intramuscular injection in BALB/c mice. pcDNA 3.1+ CFP-10 and pcDNA3.1+ ESAT-6 plasmids were prepared and defined groups of mice were injected intramuscularly with the plasmids both separately and in combination. The RNA was extracted from muscles after one month and cDNA was made using RT-PCR. The expressions of IL-4, IL-10 and IFN-gamma genes cytokines were evaluated using comparative real time PCR. Expression of IL-4 and IL-10 increased in the injection site of the mice groups which received plasmids encoding ESAT-6 and CFP-10 individually or together. More than 10-fold increase in IFN-gamma expression was found in samples taken from mice groups inoculated by plasmids encoding ESAT-6 and CFP-10 individually or together. pcDNA 3.1+ESAT-6 and pcDNA3.1+CFP-10 plasmids can increase the expression of IFN-gamma in mice after immunization.

Genomic fingerprinting of the vaccine strain of clostridium tetani by restriction fragment length polymorphism technique

Clostridium tetani or Nicolaier's bacillus is an obligatory anaerobic, Gram-positive, movable with terminal or sub terminal spore. The chromosome of C. tetanicontains 2,799,250 bp with a G+C content of 28.6%. The aim of this study was identification and genomic fingerprinting of the vaccine strain of C. tetani. The vaccine strain of C. tetani was provided by Razi Vaccine and Serum Research Institute. The seeds were inoculated into Columbia blood agar and grown for 72 h and transferred to the thioglycolate broth medium for further 36 h culturing. The cultures were incubated at 35[degree]C in anaerobic conditions. DNA extraction with phenol/ chloroform method was performed. After extraction, the consistency of DNA was assayed. Next, the vaccine strain was digested using pvuII enzyme and incubated at 37[degree]C for overnight. The digested DNA was gel-electrophoresed by 1% agarose for a short time. Then, the gel was studied with Gel Doc system and transferred to Hybond N+membrane using standard DNA blotting techniques. The vaccine strain of C. tetani genome was fingerprinted by RFLP technique. Our preliminary results showed no divergence exists in the vaccine strain used for the production tetanus toxoid during the periods of 1990-2011. Observation suggests that there is lack of significant changes in RFLP genomic fingerprinting profile of the vaccine strain. Therefore, this strain did not lose its efficiency in tetanus vaccine production. RFLP analysis is worthwhile in investigating the nature of the vaccine strain C. tetani

Isolation and purification of low molecular weight proteins from liquid culture for mycobacterium tuberculosis by chromatography

Tuberculosis [TB] is a disease caused by a bacterium called Mycobacterium tuberculosis. M. tuberculosis has different molecular weight secreted antigens. Low molecular weightproteinssecreted into the culture medium by M. tuberculosisare thought to play an important role in the development new TB diagnostic tests and new vaccines against tuberculosis. In this report, we describe isolation and purification of low-molecular-weight proteinssecreted by M. tuberculosis. Initially by biphasic medium, bacteria from Lowenstein-Jensen solid medium transferred to a Dorset-Henley liquid medium and After 6 weeks of growth, the bacteria with a 0.22 micron filters of liquid medium containing secreted proteins were isolated and the secreted proteins was precipitated by ammonium sulfate. Protein concentrations were determined by using the lowry protein assay. Then low molecular weight proteins were purified by Sephadex-G75 gel chromatography and we studied purification of low molecular weight proteins by Coomassie-Blue stained SDS-PAGE. The results showed that low molecular weight secreted proteins purified from M. tuberculosis strain DT. Also, low molecular weight proteins made up approximately 65.3% of total proteins. This study demonstrated that without break down of bacteria bodies can be purified low molecular weight secreted proteins from M. tuberculosisliquid medium by Sephadex-G75 gel chromatography

Evaluation of relative potency of human tuberculin skin test in the guinea pigs sensitized with mycobacterium tuberculosis, M. bovis BCG and M. avium

The tuberculin skin test is the most commonly used test for diagnosing tuberculosis [TB] infection. The basis of tuberculin testing is the induction of a delayed hypersensitivity reaction to the intradermal injection of tuberculin. Unfortunately, this test is incapable of distinguishing Mycobacterium tuberculosis infection from Bacille Calmette-Guerin [BCG] vaccination or infection with non-tuberculous mycobacteria. The aim of this study is to evaluate the relative potency of human tuberculin skin test] produced by Razi Vaccine and Serum Research Institute [in the guinea pigs sensitized with M. tuberculosis, M. bovis BCG and M. avium. For skin test, different groups of guinea pigs were sensitized with M. tuberculosis, M. avium and M. bovis BCG. Guinea pigs were injected intradermally with 0.1 ml of 0.4, 2 and 10 micro g/ml of tuberculin. Skin reactions [diameters of erythema, in millimeters] were independently measured 24 h after injection and results were calculated. The results showed that the specificity index of human tuberculin test for guinea pigs sensitized with M. bovis BCG in compare of guinea pigs sensitized with M. tuberculosis was equal and for guinea pigs sensitized with M. avium was not equal. This study demonstrated that human tuberculin test produced by Razi Institute for diagnosis of latent infection to M. tuberculosis has lower specificity for M. bovis in comparison with M. avium