[Genomic structure of Burkholderia mallei Razi 325, the strain used for industrial production of Mallein in Iran]

Background and Objective: Iran remains a major stronghold for glanders in the Middle East. In Iran, the non-indigenous Burkholderia mallei Razi 325 strain is used in manufacturing of the mallein, required for malleination of animals. Multi Locus Variable number tandem repeat analysis is currently the standard globally accepted genotyping system for Burkholderia mallei. This study was done to survey the genomic structure of Burkholderia mallei Razi 325, the strain used for industrial production of Mallein

Methods: In this descriptive study, a MLVA genotyping system with 4 previously-characterized loci VNTR140, VNTR1367, VNTR2065, VNTR2971 along with two new loci of VNTR24, VNTR41 was used

Results: Optimization of PCRs resulted in a single protocol that enabled simultaneous amplification of all the six loci. Sequencing of PCR products revealed there were 2, 3, 12, 6, 1 and 2 copies of the unit repeat hold in the genome of the Burkholderia mallei Razi 325 strain. This observation was extended to include the already-whole genome sequenced Chinese Burkholderia mallei ATCC 23344 and Burkholderia mallei BMQ and also Burkholderia mallei SAVP1 strains

Conclusion: The Burkholderia mallei Razi 325 strain is distinguishable from the other three strains through MLVA genotyping method

Mycobacterium tuberculosis genotyping by MIRU-VNTR method

st and ardized genotyping systems in molecular epidemiology of tuberculosis in the world. This sudy was done to determine the Mycobacterium tuberculosis genotyping by MIRU-VNTR method. Methods: This descriptive study was done on sputum, gastric lavage clinical specimens of 53 tuberculosis suspected patients. Fifty-three isolates were identified by 16S rRNAandRv-typing followed by RD typing. They were then subjected to a 12-locus [ETRA, ETRB, ETRC, ETRD, ETRE and ETRF, MIRU-10, MIRU-26, MIRU-39, MIRU-30 plus QUB-11b] MIRU-VNTR typing system. Results: In MIRU-VNTR typing, forty-four types were identified with 13 isolates classified in 4 clustered and the remaining 40 isolates representing 40 orphan patterns. In comparative analysis of MIRU-VNTR loci, MIRU-26 with 7 alleles displayed the highest diversity level [Simpson's diversity index = 0.767. Out of the 53 isolates, only one was identified as Mycobacterium bovis. All the remaining isolates were characterized as Mycobacterium tuberculosis. None of the samples was affected to Mycobacterium complex strain. No evidence of either double or co-infection of the patients with more than one species/strain was detected. Conclusion: While the genomic diversity observed by MIRU-VNTR typing sounds extensive, the population genomic structure on the whole however, seems to be homogenous. Recent transmission between studied patients does not appear to be a frequent event as only 13 isolates representing 4 MIRU-VNTR types, were assumingly epidemic

Identification of mycobacterium tuberculosis complex, using molecular methods

A high level of homogeneity observed within all bacteria in the Mycobacterium tuberculosis complex makes a property that seriously challenges traditional biochemical-based identification methods of these pathogens in the laboratory. The work presented here was conducted to characterize Mycobacterium tuberculosis complex isolates in Golestan, Northern Iran. Between 2008 and 2010, 42 mycobacterial isolates were collected from clinical tuberculosis-suspected patients in Golestan province. The isolates were sub-cultured on fresh Mycobacterium-specific culture media including glycerinated and pyruvated Lowenstein-Jensen slopes. The isolates were subsequently subjected to a PCR-based identification scheme coined Huard-Warren method. This strategy consisted of three individual algorithms namely, 16SrRNA; RV typing [Rv0577, Rv3877.8, Rvl970, Rv3120, Rvl510 and IS 1561] and RD typing [RD1, RD 4, RD9 and RD12]. All isolates were proved to be M. tuberculosis. Furthermore, none of the patients were being infected with any other member of the M. tuberculosis complex or simultaneously co-infected with two mycobacteria. This fundamental observation was independently obtained by specific culture media, RV typing and also RD typing. Considering the fact that cattle and sheep farming play an important role in the economy of the region, absence of Mycobacterium bovis in the studied isolates can be unexpected to some extent. Huard-Warren which is a simple and cost-effective identification method can be used in both reference and regional laboratory for differential diagnosis of tuberculosis

[Detection of somatic antigens in Mycobacterium avium subspecies paratuberculosis]

Johne's disease or Paratuberculosis as a chronic granulomatosis enteritis in ruminants will be caused by Mycobacerium avium subsp . Paratuberculosis . Detecting whole bacterial cell wall antigens would be helpful in potential applications for diagnosis, vaccine production, and elucidation of pathogenesis . To determine secreted somatic cell antigens of Mycobacterium avium subspecies Paratuberculosis . Standard strain [III-V] of Mycobacterium avium subspecies Paratuberculosis DNA was extracted from the cultured and gene analysis was done using PCR to confirm bacterial purity . On the other hand, protein concentrations in both media and cell entracts were determined . Furthermore, all proteins pattern were shown by SDS -PAGE . Electrophoretic findings showed some somatic antigens in the range of 19 - 100 KDa. These purified somatic antigens can be used for further study and potential application in vaccine production

Expression and purification of recombinant mycobacterium tuberculosis [TB] Antigens, ESAT-6, CFP-10 and ESAT-6/CFP-10 and their diagnosis potential for detection of TB patients

One of the most widely used methods to detect tuberculosis [TB] infection is the tuberculin skin test [TST]. The completion of Mycobacterium tuberculosis [M. tuberculosis] genome sequence has led to identification of several antigens that can be utilized for accurate diagnosis and control of TB. The aim of this study was to purify the recombinant M. tuberculosis antigens for the evaluation of their potential in TB diagnosis. The recombinant secretory antigens, ESAT-6, CFP-10 and ESAT-6/CFP-10 were produced by PCR and cloning methods. To investigate antigen specific responses of these recombinant antigens in detection of TB, ex vivo enzyme linked immunospot [ELISPOT] test in 30 clinically diagnosed TB patients was evaluated. The selected M. tuberculosis antigens were cloned, expressed and purified in Escherichia coli [BL21]. ELISPOT assay for detection of TB showed the sensitivity of 93, 90 and 100% for recombinant ESAT-6, CFP-10 and ESAT-6/CFP-10 proteins respectively, which is significantly higher than conventional TST. The recombinant antigens of ESAT-6, CFP-10 and ESAT-6/CFP-10 can be used as an accurate means of detecting TB in Iran

[Cloning, sequencing and expression of HSP70 c-terminal domain in Mycobacterium avium subsp. paratuberculosis]

Heat shock proteins [HSPs] have been shown to act as an adjuvant when co-administered with different antigens, especially tumor antigens or antigens from infectious agents. C-terminal domain of Mycobacterium tuberculosis heat shock protein 70 [Hsp70], when fused to peptide antigens, provides a unique structure that is able to induce potent immune responses. In this study, aneukaryotic expression vector pEGFP-N1, containing C-terminal domain of Mycobacterium paratuberculosis HSP 70, Green Fluorescent Protein [GFP] gene in the plasmid construct, was designed for use as a reporter. With GFP system, expression of the target protein was evaluated in the cell culture. The nucleotide sequence of the cloned gene was revealed by sequencing. The protein expression of designed plasmid was also proved by reverse transcriptase polymerase chain reaction [RT-PCR]. Our eukaryotic expression vector [pEGFP-N1 -hsp70 c-terminal] was successfully constructed and HSP70 c-terminal domain protein was expressed effectively. The current experiment, as a basis for a new DNAvaccine design, can be used for the future studies on reverse vaccinology

Outbreak of avian mycobacteriosis in flocks of domestic pigeons: an epidemiological approach

Pigeons are extensively kept for homing and racing purposes in Iran. The main objective of this study was to investigate dissemination of M. avium subsp. avium [MAA] in pigeon aviaries in Tabriz, North-western Iran. Postmortem pathologic specimens from thirty-nine out of 140 birds collected from private flocks [n = 3], were subjected to bacterial culture out of which 3-4 mycobacterial isolates were recovered. Applying a five-PCR diagnostic algorithm targeting short but definitive stretches of 16S rRNA and RV0577 genes, IS6110, IS901 and IS1245 genomic loci, proved all the isolates were MAA. They were either IS901+/IS1245+ [n = 22] or IS901+/IS1245- [n = 12]. When four healthy cattle sensitized against Mycobacterium bovis AN5 and Mycobacterium avium D4 were tuberculinated, the results confirmed the observed skin reactions against bovine tuberculin in animals sensitized with M. avium were large enough to complicate test interpretation. We believe the extent of such epidemiological impact deserves further investigation if progress in control of bovine tuberculosis is intended

[Molecular differentiation between bovine Mycobacterium bovis isolates and human Mycobacterium tuberculosis isolates in Iran through PCR-RFLP method and comparison with 5 standard strains]

Mycobacterium tuberculosis complex is consisted of homogenous organisms. They are slowly growing mycobacteria and their isolation and identification are difficult and time consuming. Differentiation of Mycobacterium bovis, causative mammalian tuberculosis, from other members of Mycobacterium tuberculosis complex is very important in epidemiology and control of disease in humans and animals. The aim of this study was to evaluate a molecular method to differentiate Mycobacteriom bovis from Mycobacterium tuberculosis. DNA human isolates of Mycobacterium tuberculosis [n=6] and Mycobacterium bovis isolates [50] were extracted and used as template in PCR. A 548bp fragment of oxyR pseudogene was amplified and digested with Alul endo nuclease. The nucleotide 285 could be adenine [M. bovis] or guanine [M. tuberculosis]. Such variation produces different restriction site for Alul. There were three incisive fragments in all Mycobacterium bovis and Mycobacterium bovis BCG strains and one incisive fragment in other members of Mycobacterium tuberculosis complex. PCR-RFLP method on 548bp fragment of oxyR gene is a rapid and accurate method to differentiate Mycobacterium bovis and Mycobacterium bovis BCG from other members of Mycobacterium tuberculosis complex

[Respiratory and intestinal cryptosporidiosis in commercial chicken in Tehran]

Materials for this research consisted 355 broiler chicken with intestinal and respiratory problems aged 11 to 45 days submitted to the department of poultry disease of Tehran Veterinary college for postmortem examination from local poultry farms. Faecal samples from cloac, cecal and intestinal contents and tracheal samples by tracheal imprints from chicken were examined for the presence of cryptosporidial oocysts after staining the samples by using modified Ziehl-Neelson technique. Cryptosporidium meleagridis was found in 8 intestinal and 3 tracheal samples. The study has brought to light the existence of chicken cryptosporidiosis in Tehran perhaps for first time