Isolation and identification of nontuberculous mycobacteria from raw milk and traditional cheese based on the 16S rRNA and hsp65 genes, Tehran, Iran.

As an important source of human food, milk can be a carrier of human pathogenic bacteria, including tuberculous and nontuberculous mycobacteria (NTM), in its raw and unpasteurized state. In this research, 175 raw milk samples and 175 traditional cheese samples were collected from traditional dairy stores in 22 regions of Tehran in a 9- month period from August 2019 to May 2020. Samples were prepared and transferred to a specialized laboratory, where they were inoculated in Lowenstein-Jensen (LJ) medium containing glycerol or sodium pyruvate, as well as Herrold's egg-yolk with and without Mycobactin J. to determine the sample's identity of samples. The recommended 16S rRNA (1436 bp) and hsp65 (644 bp) gene fragments from the positive isolates identified in Ziehl-Neelsen (Z-N) staining were amplified and sequenced using PCR and compared with the sequences of the gene fragments of reference strains available in the global GenBank database. No mycobacterial species were isolated from traditional cheese samples in microbial culture. In case of raw milk samples, a total of four bacteria were collected, all of which were found in the genetic differential testing to be NTM, including n = 1 Mycobacterium heraklionense, n = 2 Mycolicibacterium fortuitum, and n = 1 Mycobacterium thermoresistibile. The analysis of the results obtained by isolate sequencing using the 16S rRNA gene showed higher discriminatory power and percentage similarities in the identification of the isolates than the hsp65 gene.

More insights about genomic population structure of Mycobacterium avium subspecies paratuberculosis (Map) from multiple hosts in west and central provinces of Iran using a boosted genotyping approach.

To investigate the population genetic of Mycobacterium avium subsp. paratuberculosis (Map) in Iran, Mycobacterial Interspersed Repetitive Units (MIRUs) and Multi Locus Short Sequence Repeat (MLSSR) system were employed. Numerous genotypes by MIRU (N = 11) and MLSSR (N = 9) methods bearing discriminatory indices of 0.90 and 0.79 respectively, were obtained. Browsing the INRA-Nouzilly list (http://mac-inmv.tours.inra.fr/) detected 3 of the found patterns as new types. Some loci either MIRU-VNTR or SSR proved more polymorphic and therefore are recommended to be applied in priority for strain typing in the Iranian environment. While identical MIRU-VNTR or MLSSR patterns were detected among different conspecifics and geographical locations, dissimilar types were also observed at the same farms an indication of coexistence of Map strains within one herd. We suggest extension of the genotyping work described here to include more endogenous isolates in order to better analysis of transmission and virulence in epidemiology and control of paratuberculosis.

Genotyping of Mycobacterium tuberculosis complex isolated from humans and animals in northeastern Iran.

The objective of this study was to genotype Mycobacterium tuberculosis complex isolated from humans and cattle in northern Iran. Over the course of one year, a total of 120 human and 21 cattle isolates were tested using region of difference (RD)-based polymerase chain reaction (PCR) and mycobacterial interspersed repetitive unites-variable number tandem repeats (MIRU-VNTR). In M. tuberculosis, out of 120 isolates investigated, the most common genotype detected was NEW-1 (53.3%), followed by CAS/ Delhi (24.1%), Haarlem (5%), Beijing (4.16%), Uganda I (4.16%), S (3.3%), Ural (0.83%), TUR (0.83%), Uganda II (0.83%), Lam (0.83%) and Cameroon (0.83%). The HGDI rate was 0.9981 and the clustering rate was 10.83. Of the isolates, QUB26 had the highest allele diversity (h: 0.76), while the loci Mtub29 and MIRU24 had the lowest (h: 0). In M. Bovis, out of 123 collected tissue samples, 21 (17%) grew on culture media. The HGDI rate was 0.71 and clustering rate was 85.7%. The locus ETRC had the highest allele diversity (h: 0.45). The findings of this study suggest that there is high genetic diversity among M. tuberculosis isolates in Khorasan Razavi Province, which is consistent with similar results from other studies in other provinces in Iran and neighboring countries. This indicates that the prevalent genotypes in this study are spreading in the Middle East region. Furthermore, considering that M. Bovis isolates were identified in two clusters, it seems that all of them have a common origin and are circulating among the livestock farms in the province.

Molecular characterization of zoonotic Brucella species isolated from animal and human samples in Iran.

Brucellosis is an endemic infection in Iran and represents a serious health problem in humans and livestock causing important economic losses. The objective of this study was to undertake molecular characterization of Brucella spp. isolated from humans and livestock in several provinces of Iran including by multi-locus sequence typing (MLST), in order to understand the genotypes circulating in Iran and their relationship to genotypes globally. A total of 23 Brucella isolates were isolated from eight milk samples (seven cows, and one camel), human blood samples (seven), bovine lymph nodes (two), and samples from aborted fetuses (three sheep, two cows, and one goat). Phenotypic and molecular identification of Brucella isolates was performed on all isolated bacteria and showed that all were either Brucella melitensis or Brucella abortus. B. melitensis was associated with ovine/caprine and camel samples, most human isolates, and a significant minority of cattle isolates. In contrast B. abortus from livestock was associated only with isolations from bovine samples, as well as a single human sample. These results indicate that both B. melitensis and B. abortus contribute to the human brucellosis burden in Iran. B. melitensis isolates comprised three MLST-9 genotypes, the common and globally distributed ST8, a single representative of ST7, and several additional examples of ST102, a genotype previously only reported in a single isolate from a human brucellosis case believed to be acquired through travel to Iran. B. abortus isolates represented two globally common MLST-9 genotypes (ST1 and ST2), with relationships to biotype and other PCR-based typing methods consistent with previous observations. The results provide the basis for further studies examining the molecular epidemiology of Brucella circulating in Iran and the relationships of local isolates to those present globally.

Genomic pattern analysis of Burkholderia mallei field isolates by pulsed-field gel electrophoresis (PFGE) discriminatory typing.

BACKGROUND AND OBJECTIVES: Glanders is a serious zoonotic disease caused by Burkholderia mallei. Prevention, control, and treatment strategies of glanders are prerequisites for microbial source tracking. The present study was aimed to analyze the genomic pattern of B. mallei Iranian field isolates by pulsed-field gel electrophoresis (PFGE) typing. MATERIALS AND METHODS: B. mallei isolates were aerobically cultured in nutrient broth/agar supplemented with glycerol 4% for 48 h at 37°C. API 20NE identification system was used for the biochemical characterization. Genomic DNA of bacterial isolates was extracted using OIE-recommended protocol. Molecular identification of bacterial isolates was done based on amplification of BimA and IS407-flip genes. PFGE was applied to prepare the genomic pattern of B. mallei isolates. The guinea pig was used as a suitable model for studying the histopathological characterization of B. mallei. RESULTS: In both enzymatic digestion patterns by using Af1II and VspI, we found three different clonal types; І) PFGE type of B. mallei Razi 325 strain, ІІ) PFGE type of Tiger, Kordan, and Oshnavieh strains, and ІІІ) PFGE type of Semirom strain. B. mallei Razi 325 was categorized as unrelated strain which was belonged to the different cluster differing more than four bands. CONCLUSION: PFGE showed more discriminatory power and considerable reproducibility for molecular typing of B. mallei strains in our study. It is standardized the approaches for outbreak detection, pathogen phylogeny, molecular epidemiology, and population studies.

Insight into population structure of Mycobacterium tuberculosis isolates in the multiethnic province of Alborz, Iran.

BACKGROUND AND OBJECTIVES: Genetic diversity of Mycobacterium tuberculosis clinical isolates from tuberculosis patients in the multiethnic province of Alborz, Iran was assessed. MATERIALS AND METHODS: A total of 17 isolates in the period of 2012-2013 were collected and subjected to a Multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) consisted of 6 variable numbers of tandem repeats (VNTRs) including ETR-A, ETR-B, ETR-C, ElTR-D, ETR-E, ETR-F, 5 Mycobacterial Interspersed Repetitive Units including MIRU10, MIRU16, MIRU26, MIRU39, MIRU40, and 1 Queen University of Belfast locus, QUB11. RESULTS: This classified all isolates into 17 distinct MIRU-VNTR types, a reflection of a highly heterogenic population. Within the 12 used VNTR loci, ten proved highly or moderately discriminant according to the calculated HGDI scores. No cluster of isolates was identified in the study panel, giving a clustering rate of 0%, several events of SVL (N=5) and DVL (N=4) and TVL (N=3) were detected. CONCLUSION: The greater heterogeneity observed here by MLVA-VNTR analysis is most likely due to limited background data in the study region rather than a genuine more heterogeneous population compared to other provinces of the country.

Genome diversity and evolutionary characteristics of clinical isolates of Bordetella pertussis circulating in Iran.

BACKGROUND AND OBJECTIVES: The re-emergence of pertussis still is being reported all over the world. Pathogen adaptation and antigenic divergence of circulating isolates from vaccine strains are the main reasons of infection resurgence. Waning immunity is also an important factor contributing to resurgence of pertussis. MATERIALS AND METHODS: The genetic diversity and evolutionary characteristics of circulating Iranian isolates of Bordetella pertussis during February 2015 to October 2018 was investigated by pulsed-field gel electrophoresis (PFGE) and subsequently ptxA, ptxP and fim3 alleles were characterized. The next generation genome sequencing was then used to compare the genomics of ptxP1 and ptxP3 of selected isolates from PFGE dendrogram. RESULTS: PFGE differentiated 62 clinical isolates and vaccine and reference strains into 19 PFGE profiles, indicating the higher level of heterogeneity in the population during 2015-2018. The predominant B. pertussis genotype harbored pertussis toxin promoter allele, ptxP3 and the expansion of ptxA1 isolates, were also observed in our population. CONCLUSION: No changes in allelic profile of predominant clone in recent years was observed but antigenic divergence between recently circulating isolates and the vaccine strain has been progressed and significantly was higher than previous studies. The comparative genomic analysis of the ptxP3 and ptxP1 isolates indicate that changes in ptxP3 genome structure including 32 unique SNPs and three unique indels may have contributed to the expansion of the ptxP3 clone. We compared ptxP3 and ptxP1 isolates in pathogenicity-associated genes and found five of them were specific for the ptxP3 isolates. The polymorphisms in pathogenicity-associated genes suggest structural adaptations for these virulence factors.

Development of an immunoblotting assay for serodiagnosis of Burkholderia mallei infection: the whole-cell proteome-based paradigm.

BACKGROUND AND OBJECTIVES: Burkholderia mallei is the leading cause of glanders, a highly transmittable and an OIE-notifiable disease of equidae. Despite the importance of B. mallei, little is known about serodiagnosis of glanders. The present study aimed to develop an immunoblotting assay based on whole-cell proteome of B. mallei to enable accurate serodiagnosis of glanders. MATERIALS AND METHODS: Three farm horses were subcutaneously immunized with a crude suspension (106 cfu/ml) of heat-inactivated B. mallei formulated with incomplete Freund's adjuvant (IFA) to achieve a hyperimmune sera panel. The immunization was done for 1, 14 and 28 days with 1 dose of 1 ml antigen containing 106 cfu/ml. The hyperimmunity of sera was confirmed by CFT. B. mallei whole-cell proteome was prepared through sonication and the protein content was visualized by SDS-PAGE and quantified by Western blot using HRP-conjugated rabbit anti-horse IgG. A comprehensive set of positive and negative horse sera validated the test. RESULTS: A ladder pattern of the B. mallei immunoreactive antigens was seen within the region of 20-90 kDa clearly and the immunoblot was scored positive, while no reaction was seen for the negative sera. The Western blot assay indicated a noticeably higher diagnostic specificity for positive or negative sera of glanders. CONCLUSION: The whole-cell proteome-based immunoblot proved reliable and straightforward in our study. The prepared antigen was adaptable for application in immunoblotting. We assumed this improved immunoblotting system provides appropriate sensitivity and also specificity expected in serodiagnosis of glanders in endemic areas and typically in less-developed countries.

Homogeneity of Mycoplasma agalctiae vaccine strains in an agalaxy- high-burden environment.

BACKGROUND AND OBJECTIVES: Interrogation of the genomic relations between Iranian Mycoplasma agalactiae vaccine strains of Taliqan, Lorestan and Shiraz. MATERIALS AND METHODS: Two MLVA (covering VNTR loci of 5, 9, 17 and 19) and MLST (comprising dnaA, gltX, gyrB, C, tufA genes) genotyping systems plus nucleotide structure analysis of P80 gene, was conducted. RESULTS: The shared MLVA pattern represented by the three strains differed to that of the Mag PG2 laboratory strain, only at locus VNTR19 where the PG2 genome hold a 3 bp longer stretch. In MLST analysis, at dnaA, gltX, gyrB, metS and tufA loci, the three strains displayed alleles 1, 21, 2, 2 and 1, respectively. At, gltX locus a new allele (21) was detected where a new sequence type (ST33) was identified. Besides, the trio strains hold an identical nucleotide structure in their ma-mp81 gene. CONCLUSION: In explanation, lack of efficient disease control measures, has possibly contributed in evolution of a clone or a few clones that gradually overwhelmed the population over the time. Besides, the similarity between the Iranian and the PG2 strains, might be due to homoplasy or farming exercises such as animal importation. Inclusion of further local isolates in next studies will help to assess these assumptions.

Introducing the Best Six Loci in Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat (MIRU-VNTR) Typing for Mycobacterium Tuberculosis Genotyping.

BACKGROUND: Tuberculosis (TB) still remains endemic worldwide making epidemiological studies essential to mitigating efforts implicated in identifying its source, controlling, and preventing the spread of dangerous strains amongst humans such as Mycobacterium tuberculosis (Mtb). METHODS: In this study, we sought to determine the 6 Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat (MIRU-VNTR) loci with high discriminatory powers for Mtb genotyping as well as the loci with the highest and the lowest discriminatory powers for MIRU-VNTR. To conduct our search, we used several databases such as science direct, Embase (Elsevier), Web of Science, Scopus and Medline via PubMed. Searches were performed using key words including: Mycobacterium tuberculosis, MIRU-VNTR, Allele diversity, Genetic diversity and human patient. Finally, 56 articles were selected after filtering out titles, abstracts and full texts. RESULTS: Loci with high discriminatory powers included MIRU10 and MIRU26, while MIRU2, MIRU20, MIRU24 and ETRD had poor discriminatory powers. According to previous data in the literature, the loci MIRU10, MIRU26, MIRU40, QUB 26, QUB 11b and Mtub21 have high discriminatory powers. CONCLUSION: Therefore, these loci recommended for genotyping Mtb to save time and cost and to ensure the production of reliable results.

A very French connection, a brief discourse in commemoration of life and services of the late Louis Pierre Joseph Delpy on his 120th birthday anniversary.

Razi Vaccine and Serum Research Institute (RVSRI) turned 95 years old in 2015. Majority of the animal infectious diseases such as rinderpest and anthrax that used to frequently strike the historic Persia are now gone for good or under control owing to the pioneering researches conducted at the institute in the early-mid 20th century in the field of vaccine manufacturing. The earliest such scientific contributions, were truly made by the French eminent veterinarian Dr. Louis Pierre Joseph Delpy who joined the institute in 1931. In his 18 year-long directorship tenure he taught his colleagues fundamentals of vaccinology, basics of modern epidemiology, essentials of infectious disease control disciplines, the art of scientific writing and much more things that changed the institute for ever. This paper reviews the events and turning points in the first 25 years of service of the institute in a chronological way and remarks Delpy's principle involvements in all of these on the occasion of the 120 anniversary of his birth. At the entrance of the institute headquarter building where his bronze bust is placed, visitors can see a memorial etched plate that reads "... The architect of Razi and founder of Archives De L'Institute Razi (Archives of Razi Institute) was an enthusiastic scientist with a creative mind. … For the Razi community, Dr Delpy is gone but not forgotten."

Effectiveness of an inactivated paratuberculosis vaccine in Iranian sheep flocks using the Mycobacterium avium subsp paratuberculosis 316F strain.

BACKGROUND AND OBJECTIVES: Paratuberculosis (PTb) (John's disease) is an incurable chronic intestinal infection that mainly affects ruminants. PTb is caused by Mycobacterium avium subspecies paratuberculosis (MAP) with a global distribution. Despite evidences on MAP contribution in Crohn's disease its causal role is still a matter of controversy. In ruminant farming, vaccination is broadly accepted as an effective control measure of PTb. This article describes preparation and field trial of an inactivated PTb vaccine made from the MAP 316F strain. MATERIALS AND METHODS: Formulation of the vaccine was conducted based on the method traditionally used in the UK. Identity of the MAP strain was authenticated by PCR-IS900 and PCR-F57 tests. In the field, a group of 100 lambs (3-8 weeks old) were subcutaneously inoculated with the vaccine preparation under study. These animals, pre-vaccination, were all PTb ELISA negative. Serum level of antibody was determined by ELISA on days 0, 30, 60, 120 and 240, post-vaccination. RESULTS: In PCR-900 and PCR-F57, the MAP 316F strain produced two fragments of 560 and 704 bp length respectively, a confirmation of its identity as MAP bacterium. In the field trial and at the arranged time intervals, the achieved blood serum levels of antibody, attributable to the vaccine formulation, displayed considerably high values. CONCLUSION: Given that the PTb-caused economical losses in the Iranian environment are dramatically high and also the fact that future of state policy on control of PTb remains unknown, we belive vaccination of animals is the best recommendable practice.

Genome Sequence of Pasteurella multocida Razi 0002 of Avian Origin.

We report here on the genome sequence of Pasteurella multocida Razi 0002 of avian origin, isolated in Iran. The genome has a size of 2,289,036 bp, a G+C content of 40.3%, and is predicted to contain 2,079 coding sequences.

Genome Sequence of Pasteurella multocida Strain Razi_Pm0001.

We report here the genome sequence of Pasteurella multocida Razi_Pm0001 from bovine origin, isolated in Iran in 1936. The genome has a size of 2,360,663 bp, a G+C content of 40.4%, and is predicted to contain 2,052 coding sequences.

First Report of Mycobacterium bovis Isolation from a European Fallow Deer (Dama Dama Dama) in Iran.

At present, most of Iran is free of bovine tuberculosis (TB). The strategy of control and eradication in Iran involves a tuberculation test and slaughter of reactors, a procedure transformed the present-day prevalence of TB into a sporadic occurrence. This paper describes the first report of bovine tuberculosis in a European fallow deer (Dama dama dama) in Iran. The deer was emaciated and found dead in the Hoveize Provincial Zoo Park. Post-mortem examinations revealed multifocal granulomatous and suppurative abscesses in the lungs and mesenteric lymph nodes. These post-mortem indicators led the authors to suspect TB, and the PCR test and bacteriology tests confirmed it as an infection by the Mycobacterium bovis. This survey discusses the important implications of such findings for wildlife, especially livestock, as well as for human TB disease control, because deer are often conserved in public zoos and humans often come into contact with them.

Molecular typing methods used in studies of Mycobacterium tuberculosis in Iran: a systematic review.

BACKGROUND AND OBJECTIVES: Molecular typing methods are important and useful tools to assess the transmission, diversity of strains and differentiation between new infections and relapses which can effectively help in controlling infections. The aim of this study was to evaluate the molecular typing methods which have been used in Iran. By evaluating the results and discriminatory power of each method, we can assign appropriate weight to each technique and ultimately offer a common strategy for future epidemiological studies. METHOD: We searched several databases to identify studies addressing Mycobacterium tuberculosis molecular epidemiology in Iran. Hunter-Gaston discrimination index (HGDI) was used to evaluate the discriminatory power in each method. Relevant articles were selected and analyzed; HGDI index was calculated for each technique. RESULTS: The most common genotyping methods used in the articles were RFLP, MIRU-VNTR, spoligotyping, PFGE and RAPD-PCR. The most frequently techniques were IS6110-RFLP, MIRU-VNTR and spoligotyping alone or in combination. The highest discrimination power (average HGDI: 0.9916) was obtained by RFLP followed by MIRU-VNTR (average HGDI: 0.9638) and spoligotyping (average HGDI: 0.9041) respectively. CONCLUSION: Combination of MIRU-VNTR with spoligotyping can be recommended for large-scale genotyping in Iran. It seems appropriate to consider spoligotyping as the first technique for screening followed by other techniques with higher discrimination power such as MIRU-VNTR or IS6110-RFLP.

Frequency of mycobacterial infections in retailed fish in Karaj: An Iranian perspective.

OBJECTIVE/BACKGROUND: Mycobacterium species comprise one of the most frequently-reported causative agents in granulomatous lesions of fish. Also, mycobacteria have been documented as fish-born zoonoses. Ornamental aquaria, commercial aquaculture, and wild fisheries therefore produce a potential risk of infection for humans through physical contact and consumption of fishes. A number of fish mycobacteriosis and fish-born zoonotic cases have been reported to date in Iran. METHODS: In order to examine the frequency of mycobacteria existence in fish supplied to the public in seafood retail outlets, whole fresh fish from cold water (n=50) and tropical (n=50) audible fish were obtained from five stores in Karaj, the central city of Alborz province. The head, tail, and offal of these fish were sampled during the necropsy and used for mycobacterial culture on Lowenstein-Jensen slopes. RESULTS: In total, 15 acid-fast isolates were collected including 10 from tropical fish. The 16S ribosomal RNA and hsp genes of all isolates were polymerase chain reaction amplified and sequenced. Mycobacterium fortuitum was the most frequent mycobacterium identified in the study panel according to the Basic Local Alignment Search Tool search results. Work is still ongoing to characterize the other cultured mycobacterial isolates. CONCLUSION: The detection of 15 culturable mycobacterial isolates from 100 audible fish is an indication of bacteria highly active in the colonization in fish populations. Assessing the potential zoonotic risk raised from exposure of human to fishes in Iran demands search for evidence of linkages between mycobacteria infecting human cases and fishes.

Interferon-γ assay, a high-sensitivity, specific and appropriate method for detection of bovine tuberculosis in cattle.

Bovine tuberculosis (TB) is an important zoonotic disease that is caused by Mycobacterium bovis. Eradication efforts in developed countries have reduced the prevalence of this disease significantly. TB can be difficult to diagnose based only on the clinical signs; therefore, it is usually diagnosed in the field with the tuberculin skin test and diagnostic blood tests, including the lymphocyte proliferation assay, the interferon (IFN)-γ assay, and enzyme-linked immunosorbent assay. The aim of this study was to compare the tuberculin and IFN-γ tests. A total of 110 animals were evaluated by tuberculin skin test (TST) and IFN-γ assay; the culture was selected as a gold standard. The animals were selected randomly from 700 cattle on dairy farms, aged 3-5years and suspected of having TB. Ten cattle were positive using the TST and nine were positive by IFN-γ assay. All nine positive samples in the IFN-γ assay were positive in culture too. The observed errors in IFN-γ assay were less due to laboratorial tools. It is suggested that all positive samples in TST are also positive by IFN-γ too.

Genotyping analysis of bovine, ovine, and caprine paratuberculosis in Iran: An IS900-RFLP study.

OBJECTIVE/BACKGROUND: Mycobacterium avium subspecies paratuberculosis (MAP) has been reported in Iranian cattle since 1965. Little is known on the population genetics of MAP in this Middle-Eastern State. A principle scope of this study was therefore genetic characterization of MAP isolates collected from farm animals in Iran. METHODS: Sixteen field isolates of MAP collected from bovine (n=8), ovine (n=3), and caprine (n=2) hosts in Alborz, Fars, Isfahan, Qazvin, Tehran, and Zanjan provinces were subcultured on mycobactin J-supplemented Herrold's egg slopes in order to provide the required genomic material. The laboratory strain MAP III & V was included as the reference strain. IS900-RFLP (Restriction Fragment length Polymorphism) was conducted using BsteII restriction enzyme. Application of IS900-RFLP genotyping on 16 Iranian MAP isolates in the present study classified them into six observable but similar types represented by two clustered and four orphan types. The laboratory strain MAP III & V displayed a totally different pattern easily distinguishable from that of Iranians. RESULTS: Detection of six genotypes among 16 wild isolates is an indication of a population with a potentially high level of diversity. We assume that, with inclusion of more field isolates, it is very likely that even higher diversity may be observed within the studied isolates. The different patterns displayed by the Iranians and the laboratory strain in this work might explain the independent evolutionary pathways these have gone through to evolve from their ancestral clones. CONCLUSION: Further description on population genetic of MAP in Iran urges for more epidemiological work using similar and alternative standard genotyping system.

Rapid identification of Mycobacterium avium ssp paratuberculosis laboratory strains by IS900-Nested polymerase chain reaction.

INTRODUTION: Mycobacterium avium ssp paratuberculosis (MAP) causes paratuberculosis (Johne's disease) in ruminants. As a species, M. avium comprises M. avium subsp. hominissuis and a number of clones that are known to have evolved from this subspecies, namely M. avium subsp. avium (MAA), M. avium subsp. silvaticum, and MAP. Despite the very high genomic similarity of MAP and MAA, the insertion sequence IS900, which is 1,451-bp long, is now understood to be exclusively present in 10-20 copies in the genome of MAP. In the present study, a multidiscipline polymerase chain reaction (PCR)-based algorithm targeting16SrRNA, IS6110, IS901, IS1245, and IS900 markers has been employed to differentiate between six laboratory strains of M. avium complex (including MAP 316F, III&V, and 2e plus MAA D4), Mycobacterium tuberculosis DT, and Mycobacterium bovis AN5 strains used at the Razi Institute (Tehran, Iran) for the preparation of paratuberculin, avian, human, and bovine tuberculin, respectively. MATERILS AND METHODS: Three laboratory strains of III&V, 2e, and 316F were subcultured on Herrold's egg yolk medium, whereas the MAA strain of D4 along with M. bovis AN5 and M. tuberculosis DT were subcultured on Lowenstein-Jensen slopes. All the inoculated culture tubes were incubated for 8weeks at 37°C. Eventually, their genomic DNA was extracted according to the method of van Soolingen. Five individual PCRs were conducted on these templates to amplify 16SrRNA (genus-specific marker shared by all mycobacteria), IS900 (MAP-specific marker), IS901 (MAA-specific marker), IS1245 (M. avium complex (MAC)-specific marker), and IS6110 (M. tuberculosis complex (MTC)-specific marker) loci. RESULTS: Consequently, a 543-bp amplicon was amplified by all the six strains in PCR against 16SrRNA, an indication of their identity as members of Mycobacterium genus. A 245-bp fragment was detected in only IS6110-PCR with M. bovis AN5 as well as M. tuberculosis DT. In the IS1245 assessment, the MAA strain of D4 produced a 427-bp amplicon, whereas none of the other studied strains produced this amplicon. A 1,108-bp amplicon fragment of the IS901 marker was successfully produced by MAA strain, whereas no PCR product was achieved in amplification of all the three MAP strains. In IS900-nested PCR, the three MAP strains produced the expected 400-bp and 298-bp fragments CONCLUTION: However, no amplification was observed with other strains. Two main achievements of this work are the development of an efficient means of differentiation between the six Razi laboratory mycobacterial strains and characterization of the genomic profile of these strains, a capability that is vital when cross contamination is potentially an important concern.