ABSTRACT Background and Aims Tuberculosis (TB) remains a major global health problem and poses significant challenges for diagnosis and treatment. The World Health Organization (WHO) recommends the use of molecular methods for the diagnosis and genotyping of Mycobacterium tuberculosis complex (MTBC). Mycobacterial interspersed repetitive unit‐variable number tandem repeats (MIRU‐VNTR) has proven to be a valuable method for studying the genetic diversity of Mycobacterium tuberculosis (MTB). To the best of our knowledge, no previous study has investigated MTB diversity in Ilam, west of Iran. Therefore, this study aimed to investigate the genetic diversity of Mycobacterium tuberculosis isolates and the occurrence of rifampicin‐resistant Mycobacterium tuberculosis (RR‐TB). Methods In total, 643 suspected cases of TB were collected from March 2022 to November 2023. Acid‐fast bacilli (AFB) were detected using Ziehl–Neelsen (ZN) staining, fluorochrome staining, and culture on Löwenstein–Jensen medium. GeneXpert MTB/RIF method was applied to detect MTB and RR‐TB. Finally, 15‐locus‐based MIRU‐VNTR was used for molecular genotyping. Results Out of 643 specimens, 20/643 (3.11%) specimens were diagnosed as positive for MTB by culture. GeneXpert MTB/RIF confirmed RR‐TB in one sample. Positive isolates were placed in nine different clusters using MIRU‐VNTR genotyping. All isolates were not assignable to the sublineages in the MIRU‐VNTRplus database but were close to the Delhi/CAS. Conclusion The high clustering rate in our results indicates that TB transmission in this region originates from multiple sources and reflects active transmission rather than reactivation of latent TB. In addition, the lineages of MTB isolates identified in Ilam were closely related to those reported from other Iranian provinces and neighboring countries, indicating epidemiological links. Improving diagnostic capacity and implementing effective control measures are essential to reduce TB transmission.

Shigella is a highly invasive pathogen that causes dysentery and is associated with significant morbidity and mortality in children under five years of age. This agent is a major public health problem in developing countries. Multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) is a reliable, cost-effective typing method with high discriminatory power and reproducible results. The rise of drug resistance in Shigella strains is a growing global health threat. Despite the significance of Shigella in Iran, there is limited knowledge about genetic diversity and drug resistance profiles of local strains. Therefore, the purpose of this study was to characterize the genetic diversity and drug resistance profiles of Shigella strains isolated in Ahvaz, Iran. A total of 49 Shigella flexneri isolates were recovered from 500 stool samples of pediatric patients. Routine biochemical tests were used to identify all isolates. Antimicrobial susceptibility testing was performed, and resistance genes were detected by polymerase chain reaction (PCR). Extended-spectrum β-lactamases (ESBL), carbapenemase, and Metallo-β-lactamase (MBL) production were detected phenotypically using combination disk assays and confirmed by the CLSI-recommended modified Carbapenem inactivation method (mCIM) and EDTA-modified carbapenem inactivation method (eCIM). MLVA based on seven VNTR loci was performed to characterize the genetic diversity of the isolates. All 49 isolates were resistant to ceftazidime, trimethoprim/sulfamethoxazole, ampicillin, and ceftriaxone (100% each). High resistance rates were also observed for imipenem 36/49 (73.5%), meropenem 36/49 (73.5%), azithromycin 21/49 (42.9%), and ciprofloxacin 16/49 (32.7%). Furthermore, phenotypic testing revealed ESBL production in 46/49 (93.9%) isolates and carbapenemase activity in 36/49 (73.5%), of which 22/49 (44.9%) were MBL. PCR analysis identified blaCTX-M 38/49 (77.6%) and blaSHV 35/49 (71.4%) as the most prevalent ESBL genes, whereas blaNDM 14/49 (28.6%), and blaOXA-48 14/49 (28.6%) were the most common carbapenemase genes. MLVA typing divided the isolates into 22 different MLVA types, including 10 clusters and 12 singletons, and locus ms21 showed the highest discriminatory power. The isolates exhibited high genetic diversity with a non-clonal distribution of resistance, which indicates dissemination through horizontal gene transfer. Our results demonstrated that mCIM/eCIM and MLVA are viable methods for investigating Shigella species as they are cost-effective, provide quick results, and allow for easy sharing of numerical data between laboratories.

BACKGROUND:Escherichia coli O157 is a major foodborne pathogen associated with severe human disease, with cattle serving as its primary reservoir, and in this study multilocus variable-number tandem-repeat analysis (MLVA) was applied to investigate the genetic diversity, distribution, and population structure of isolates collected in Fars Province, Iran. METHOD:This study was performed on 150 swab samples were collected from rectum and carcasses of cattle, sheep and goats and fruit juice in Fars province. Samples were processed following standard bacteriological procedures. MLVA method was performed based on polymerase chain reaction (PCR) by using four variable-number tandem repeat (VNTR). RESULTS:22 isolates were identified as E. coli O157:H7 that 15, 6 and 1 were from cattle, sheep and fruit juice, respectively. By using MLVA technique, 22 isolates were separated into 15 distinct MLVA types. MST algorithm showed five genetically related clusters and by UPGMA method isolates were classified in 3 main cluster and 8 sub clusters. CONCLUSION:This study showed that MLVA can be used as a valid, inexpensive and rapid method to trace and analyze the phylogenetic relationships between E. coli strains in epidemiological research.

Pasteurella multocida is a zoonotic bacterium commonly found in the oropharyngeal microbiota of cats; despite its clinical relevance, molecular data on feline isolates in Iran remain limited. This study aimed to provide molecular and phenotypic characterization of P. multocida isolates obtained from feline sources in Iran. This study characterized 18 P. multocida isolates recovered from oral swabs of cats previously sampled in Ahvaz, Iran. After polymerase chain reaction (PCR) confirmation targeting the kmt1 gene, multilocus sequence typing (MLST) was performed using the Rural Industries Research and Development Corporation (RIRDC) scheme. The MLST analysis assigned the 18 isolates to 14 distinct sequence types (STs). High genetic diversity was observed, with no ST dominating the population. Our results identified several STs, including ST30, ST344, ST487, and ST551, that have been reported worldwide, highlighting their epidemiological relevance. This study provides the first MLST data for feline P. multocida in Iran and contributes to global understanding of the species' genetic diversity.

Clostridium perfringens is a component of the normal gut microbiota in humans and animals. However, it can become pathogenic under certain conditions, causing serious intestinal and systemic diseases. Enterotoxaemia is one of these diseases causing significant mortality in livestock annually. Analysis of genetic diversity or relatedness of isolates with molecular typing methods can be helpful for understanding the epidemiology of these infections and observing population structures of these bacteria. The aim of this study was to perform molecular typing of Clostridium perfringens isolates obtained from both healthy and clinically suspected enterotoxemic livestock in Fars Province using Multiple Locus Variable Tandem Repeat Analysis (MLVA) method. Collection of previously isolated and preserved Clostridium perfringens along with new collected isolates from healthy and diseased livestock were evaluated for toxin type and some virulence genes including cpe, tpeL, netB, and β2. Genetic typing of isolates was done using specific primers for amplification of eight Variable Number Tandem Repeat (VNTR) loci. PCR was done and electrophoresis banding patterns of fragments were analyzed by BioNumerics software. The results of typing revealed 71 distinct MLVA profiles from 74 strains. The strains analyzed included 27 clinical isolates, 43 non-clinical isolates, three vaccine strains, and the reference strain 13 obtained from the GenBank database. Clostridium perfringens strain 13 sequence was used as a reference strain and was evaluated for comparison of sequences for each locus. Clustering of isolates according to the minimum spanning tree showed 5 different clusters, excluding three orphan isolates. Clinical and non-clinical isolates were scattered in all clusters and no defined cluster related to health status, date of isolation, kind of host and place of isolation was detected. The total diversity index of our typing method was estimated to be 0.99. Overall MLVA molecular typing method shows high genetic diversity among Clostridium perfringens isolates.

This tribute, written as a brief pictorial essay, a biographical sketch, and perhaps a glimpse into his personality, is intended to stimulate an appreciation for the remarkable veterinarian Dr. Morteza Kaveh Kachouie, for the services he provided and the legacy he left for his country. Being indefatigable in his commitment to promote public health and reduce social disparities in his society, Dr. Kaveh remains among the few we know today who have made history in their contributions to the Hessarak (Karaj) people, to the veterinary field, and beyond.In today''s fast-moving world, where non-stop advances in technology overwhelm all aspects of our lives, the priceless experiences of people who came before us are easily undermined or overlooked. Writing biographies, therefore, can compensate by paying respect to our luminaries and inspiring the young generations.On January 9, 2025, under the auspices of the Ministry of Agriculture of Iran, a prestigious ceremony hosting several senior State figures including MPs, the under-secretary of State, Minister of Agriculture, Academy of Science members, university professors, veterinarian and non-veterinarian officials plus many other professionals from government and private sector, was held in Tehran to mark the 100th anniversary of the Razi Vaccine & Serum Research Institute. The event acknowledged the institute''s outstanding achievements at the national and regional scale in the development of knowledge and know-how in the field of manufacturing vaccines and other biologicals with therapeutic/diagnostic application, and also the diagnosis of animal infectious diseases. In the centenary of Razi Institute''s birth, it is highly appropriate to pay tribute to the institute''s giants. Between 1985 and 1990, Razi witnessed demise of two of its legendary figures Dr Aziz Rafie and Dr Morteza Kaveh Kachouie, the second and the third directors of the institute succeeding the late Dr Delpy. Together, these three men led Razi for nearly half a century. Dr Kaveh, whom we know more for his public benefit activities, passed away on 10th December 1987, aged 81, in Tehran.This article is a brief tribute to the memory of Dr. Kaveh for his lifelong commitment and dedication to making a big difference.

The multiplicity of infection sources along with the genetically homogenous nature of Mycobacterium avium subsp. paratuberculosis (MAP) necessitates the application of high-discriminative fingerprinting techniques to establish epidemiological links and control of paratuberculosis in ruminants. The present study aimed to develop a simple Multiple-Locus Variable-Number Tandem-Repeat Analysis (MLVA) assay suitable for low-budget laboratory settings. A bioinformatics screening was conducted on three annotated MAP genomes (K10, MAP4, and MAP-JIII-386) leading to the detection of 7 potential polymorphic variable-number tandem repeats (VNTRs). A MAP collection of the three aforementioned strains, 30 field isolates and 2 laboratory strains from Iran plus 59 global Mycobacterium avium complex strains were subsequently genotyped at laboratory or in situ. Findings were validated against the French INRAE MLVA typing system (Thibault’s scheme) dedicated to typing Mycobacterium avium subsp. paratuberculosis, hominissuis, avium, and silvaticum. Fourty-one MLVA types were detected by our suggested MLVA scheme, compared to 45 types found by Thibault’s scheme. The calculated discriminatory power of the MLVA for the former and the latter was 0.93 and 0.95, respectively. The suggested MLVA scheme discriminated between isolates with identical types determined by Thibault’s scheme. Noting the fact that strains grouped in INMV types 2, 3, 4, 5, 33, 39, 68, 82, 131, 219, 220, New1 and New 25 were further resolved into sub-groups, we suggest considering VNTR 1067 (DI = 0.71), MAP 4356 (DI = 0.68), MATR 7 (DI = 0.53), MAP 3824 (DI = 0.56) and MATR 6 (DI = 0.49), in any future upgrading of the current version of the Thibault’s scheme.

In 1925, the Razi Vaccine and Serum Research Institute was established due to the rinderpest outbreak in Iran. This institute, which is currently located in the city of Karaj, has various departments for the production of biological products for humans, livestock, and poultry, research departments in the field of developmental research and basic and applied veterinary research, diagnostic departments for diagnosing diseases and isolating pathogens, as well as educational departments for holding domestic, national and international courses and also holding various postgraduate education levels.Currently, the Razi Vaccine and Serum Research Institute is on the verge of its centenary, and during a century of activity and service to the community''s health, the institute''s researchers have provided valuable services in line with its organizational duties.The mission of the Razi Institute is to contribute to the public health of the human and livestock communities of the country through the eradication and control of livestock, poultry and human diseases by producing biological products, veterinary research and research and diagnosis of many animal and common diseases and the isolation of pathogenic agents in specialized laboratories and national and international reference laboratories, publishing reputable international journals, expanding production and research throughout the country through branches of this institute throughout Iran, controlling the quality of all domestic and imported biological products at the institute and country levels, holding training courses, seminars and training students at educational levels, research and scientific research on biological products and related topics, and producing technical knowledge at the institute, country and regional levels which has served well in these matters over these many years.

Background: Shiga toxin-producing Escherichia coli (STEC) strains are bacteria that cause gastrointestinal illnesses in humans. The strain E. coli O157:H7 diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome in humans. Cattle and small ruminants are the main reservoirs of E. coli O157:H7. When shed in animal faeces, the bacteria can contaminate soil, water and food. Aim: We genotyped bacterial isolates and identified their genetic associations and source of origin in order to reduce the spread of infections and improve disease control. Methods: An Enterobacterial Repetitive Intergenic Consensus (ERIC) polymerase chain reaction (PCR) was used to determine the genetic relationships and potential cross-contamination among isolates. The genetic fingerprint patterns obtained from the isolates were compared to determine their genetic associations. A total of twenty-two E. coli O157:H7 isolates were identified from samples collected in carcasses and rectums of cattle, sheep, goats, and from fruit juice samples. SMAC agar supplemented with specific nutrients was used to selectively and differentially isolate E. coli O157:H7. The isolates were then subjected to ERIC-PCR using ERIC1 and ERIC2 primers. Results: The genetic relationships and potential cross-contamination among isolates were evaluated using the fingerprint patterns obtained. Their similarities ranged from 46% to 100%. Isolates from different hosts and sources exhibited similar patterns, indicating a high prevalence of specific clones in various hosts. The study revealed the genetic diversity of E. coli O157:H7 collected from identical sources. Conclusion: This study highlights the importance of genotyping and identifying the sources of E. coli O157:H7 strains. Understanding the genetic relationships and potential cross-contamination among isolates can help prevent the spread of infections and improve the efficacy of disease control.

Abstract Contagious agalactiae is a disease affecting small ruminants, caused by the bacterium Mycoplasma agalactiae. It is characterized by mastitis, polyarthritis, and keratoconjunctivitis. In regions heavily reliant on animal husbandry, this disease can result in substantial economic losses. Effective prevention methods include vaccination and accurate disease diagnosis in sheep and goats. The Surface lipoprotein P48 of M. agalactiae is constantly expressed and considered an immunodominant antigen present in all isolates. Antigenic variation is the primary cause of sensitivity differences in the p48-based serum assay. Recent studies have revealed fundamental differences in the p48 protein of Iranian isolates compared to previous ones. Therefore, rp48d protein was expressed in Escherichia coli BL21 (DE3) cells and purified using Ni-resin through an affinity batch formation method. Experimental sheep were divided into four groups for immunization: rp48d, RVSRI agalactiae vaccine, and two control groups (adjuvant and non-vaccinated control). Additionally, one hundred blood serum samples taken from vaccinated and non-vaccinated sheep (Fashandi breed) from various herds in the Taleghan region were analyzed using indirect ELISA. The results demonstrated that 97.50% and 100% sensitivity and specificity, respectively in rp48d-ELISA. Based on our findings, we can conclude that the in-house rp48d-ELISA is highly effective in detecting specific antibodies in vaccinated or infected sheep. This is due to the significant protein similarity between the structure of the rp48d protein utilized in the design of the ELISA test and mycoplasma agalactiae circulating in the region and used in vaccine production.

Johne's disease (JD) affects domestic and wild animals across the globe. Paratuberculosis exerts huge economic impacts on the animal industry. Despite significant economic losses, little knowledge is available on the epidemiological status of Paratuberculosis in the animal population of Iran. The present study aimed to evaluate the prevalence rate of this disease in the Iranian animal population with confidence interval (CI) and p-value. The search was conducted on and screened the electronic international and national databases. Thereafter, sufficient and relevant data were extracted. Data were analyzed in STATA software (version 14). Prevalence disease rates were determined using random effect models. A total of 52 articles were included in the systematic review. According to the results, the overall disease incidence rate in Iran was 20.39%. The prevalence rate of JD was 22.33% (95% CI, 18.87-25.78) in the cattle population and 25.61% (95% CI, 21.43-29.78) in sheep. This study pinpointed that cattle and sheep were the most commonly infected hosts. The highest prevalence rate of disease was 35.88% in Tehran (95% CI, 16.77-54.99), followed by 32.86% (95% CI, 25.07-40.65), and 20.10% (95% CI, 14.63-25.58) in Khorasan Razavi and Kerman, respectively. The lowest prevalence rate of JD was 2.27% in Ilam (95% CI, 0.84-3.70). Based on this result, molecular-based methods were properly compared to other diagnostic methods. This study reported Mycobacterium avium subsp. paratuberculosis (MAP) prevalence in dairy herds in the provinces of Iran. The infection transmission from animal sources to humans and the potential role of MAP in human disease highlight a critical need for further study on this issue.

Non-tuberculous mycobacteria (NTM) are among the most important pathogens in wild, captive, marine, and freshwater fish species. So, it is important to consider fish as the primary source of infection for aquarium fish and humans. The present study analyzed the occurrence of NTM in aquarium fish in Ilam, west of Iran. In total, 50 samples of infected fish were collected from different aquariums. Following initial sample processing, sediment of each sample was inoculated into Lowenstein-Jensen and Herrold egg media. The positive colonies were investigated with, growth rate, pigmentation, colony morphology, niacin accumulation, nitrate reduction, catalase activity, urease activity, and arylsulfatase activity. Also, molecular identification was carried out by sequencing of heat shock protein 65 kD gene (hsp65) sequence analysis. According to our results, NTM were isolated from 13 samples (26%), comprising 6 (46.2%) rapid growing, and 7 (53.8%) slow growing mycobacteria. In addition, Mycobacterium marinum was the most common NTM isolated in ornamental fish, which is potentially dangerous for both fish and humans. In conclusion, the current study indicates that ornamental fish play a significant role as a source of NTM

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.

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.

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.

Background Tuberculosis (TB) is an old issue that is presently measured as a significant challenge. Molecular characterization of Mycobacterium tuberculosis has contributed to TB control. The present survey was aimed to assess the genetic diversity of M. tuberculosis strains isolated from patients with TB in Karaj, Iran. Methods Seventeen M. tuberculosis isolates from 2012 to 2013 were collected and subjected to an IS6110 restriction fragment length polymorphism (IS6110-RFLP) analysis. Demographically, 6 females and 14 males who had Iranian citizenship were included in this study. Results Sixteen different genetic types were obtained after enzymatic digestion and RFL analysis. Copy numbers of IS6110 in each isolate ranged from 0 to 12. The majority of isolates (66%) harbored copy numbers between 6 and 12. Each isolates harbored 6.9 copies of the IS6110 marker. Nine isolates harbored 10 to 12 copies of the IS6110 marker, 5 isolates harbored 6 to 10 copies, and 2 others harbored copies less than 6. No copy of IS6110 was found among the 4 isolates. No relationship was found between gender and copy numbers. Conclusions The high genetic diversity found amongst the M. tuberculosis isolates maybe show different sources of infection and the importance of reemerging of the TB. However, further surveys should perform to assess other molecular epidemiologic aspects of M. tuberculosis 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.

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.

Aims and objectives: Mycobacterium avium subspecies paratuberculosis was first diagnosed and reported in imported cattle from England during 1962-1965. Further studies have shown that paratuberculosis is scattered throughout Iranian ruminants population. In spite of heavy economic loses; little genetic information is available. So, present study was carried out in order to obtain a better understanding of the MAP genetic diversity in Iranian isolates. Methods: During a 7-month period, 36 isolates of MAP was collected from milk and feces obtained from sheep, goat and cattle originating 10 provinces plus a further eleven archived MAP isolates and two MAP vaccine strains (MAP 316F & III &V) and two D4 reference strains (Weybridge and Turkish) were included and digested by restriction endonucleases PstI. Digested DNA was hybridised with a standard IS900 probe. Genomic fingerprints were scanned by camera and analysed using the software Gel pro analyzer & Bionumerix software package version 6.5 (Applied Maths Belgium). Dendrogram and Minimum Spanning Tree was draw using arithmetic averages (UPGMA) for Iranian isolates and vaccine strains. Results: ten RFLP (Pst I) types were detected and designated as B (bovine, ovine and caprine), and G (bovine, and caprine) and eight new type (three of them singletons and five cluster) in accordance with the study by pavlik et al 1999. The largest cluster (G strain) contained 45.8% of Iranian isolates (n = 22) then type II (B strain), represented by 11 isolates, recovered from all the three animals in six provinces. A single type was identified only in cattle from three provinces. The identical RFLP type was shown by MAP vaccine strains were different from the Iranian isolates. Two D4 reference strains had restriction pattern but did not hybridize to IS900 sequence. Dendrogram result showed B profiles close to G profile and MAP vaccine strains profiles (>98). The results of Minimum Spanning Tree also showed that all of the clusters and orphan were derived directly or indirectly from B profile with multiple hosts throughout the country. This pattern has been seen from throughout the world and multiple hosts. Conclusions: RFLP analysis showed that the Iranian isolates have perhaps originated from B specific profile but it still need to more future works using genotyping methods.

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.