Molecular investigation of virulence factors of Brucella melitensis and Brucella abortus strains isolated from clinical and non-clinical samples

Accepted Manuscript Molecular investigation of virulence factors of Brucella melitensis and Brucella abortus strains isolated from clinical and non-clinical samples Reza Mirnejad, Faramarz Masjedian Jazi, Shayan Mostafaei, Mansour Sedighi PII:

S0882-4010(17)30468-0

DOI:

10.1016/j.micpath.2017.05.019

Reference:

YMPAT 2263

To appear in:

Microbial Pathogenesis

Received Date: 29 April 2017 Revised Date:

6 May 2017

Accepted Date: 11 May 2017

Please cite this article as: Mirnejad R, Jazi FM, Mostafaei S, Sedighi M, Molecular investigation of virulence factors of Brucella melitensis and Brucella abortus strains isolated from clinical and non-clinical samples, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2017.05.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Original Article

Molecular Investigation of Virulence Factors of Brucella melitensis and

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Running Title: Virulence Factors of B. melitensis and B. abortus

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Brucella abortus Strains Isolated from Clinical and Non-clinical Samples

a

Molecular Biology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran

Department of Microbiology, school of Medicine, Iran University of Medical Sciences, Tehran, IR Iran c

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Department of Biostatistics, school of Medical Sciences, Tarbiat-Modares University, Tehran, IR Iran

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d

Researcher in the Rheumatology Research Center, Tehran University of Medical Sciences, IR Iran

* Corresponding Author: Mansour Sedighi, Department of Microbiology, school of

Medicine, Iran University of Medical Sciences, Tehran, IR Iran. Tell: (0098) 936-690-5766,

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b

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Reza Mirnejad a, Faramarz Masjedian Jazi b, Shayan Mostafaei c, d, Mansour Sedighi b, *

Postal Code: 1439914153, E-mail: [email protected]

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Address of Authors: 1- Reza Mirnejad: PhD, E-mail: [email protected]

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2- Faramarz Masjedian Jazi: PhD, E-mail: [email protected] 3- Shayan Mostafaei: MsC, E-mail: [email protected]

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4- Mansour Sedighi: MsC, E-mail: [email protected]

Acknowledgement:

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The authors are greatly thankful to Saadat Pirouzi for help with the English language version of this paper. Also, authors would like to thank the director and principal of Iran university of

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medical sciences for their constant encouragement and support for the current study.

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Molecular Investigation of Virulence Factors of Brucella melitensis and

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Running Title: Virulence Factors of B. melitensis and B. abortus

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Brucella abortus Strains Isolated from Clinical and Non-clinical Samples

Reza Mirnejad a, Faramarz Masjedian Jazi b, Shayan Mostafaei c, d, Mansour Sedighi b, *

Department of Microbiology, school of Medicine, Iran University of Medical Sciences, Tehran, IR Iran c

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Department of Biostatistics, school of Medical Sciences, Tarbiat-Modares University, Tehran, IR Iran

* Corresponding Author: Mansour Sedighi, Department of Microbiology, school of

Medicine, Iran University of Medical Sciences, Tehran, IR Iran. Tell: (0098) 918 374 9460,

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d

Researcher in the Rheumatology Research Center, Tehran University of Medical Sciences, IR Iran

Postal Code: 1439914153, E-mail: [email protected]

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b

Molecular Biology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran

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a

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ABSTRACT Brucella is zoonotic pathogen that induces abortion and sterility in domestic mammals and chronic infections in humans called Malta fever. It is a facultative intracellular potential

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pathogen with high infectivity. The virulence of Brucella is dependent upon its potential virulence factors such as enzymes and cell envelope associated virulence genes. The aim of this study was to investigate the Brucella virulence factors among strains isolated from humans and

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animals in different parts of Iran. Seventy eight strains of Brucella species isolated from suspected human and animal cases from several provinces of Iran during 2015-2016 and

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identified by phenotypic and molecular methods. The multiplex-PCR (M-PCR) assay was performed in order to detect the ure, wbkA, omp19, mviN, manA and perA genes by using gene specific primers. Out of 78 isolates of Brucella spp., 57 (73%) and 21 (27%) isolates were detected as B. melitensis and B. abortus, respectively, by molecular method. The relative

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frequency of virulence genes ure, wbkA, omp19, mviN, manA and perA were 74.4%, 89.7%, 93.6%, 94.9%, 100% and 92.3%, respectively. Our results indicate that the most of Brucella strains isolated from this region possess high percent of virulence factor genes (ure, wbkA,

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omp19, mviN, manA and perA) in their genome. So, each step of infection can be mediated by a number of virulence factors and each strain may have a unique combination of these factors that

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affected the rate of bacterial pathogenesis. Keywords: Brucella melitensis; Brucella abortus; Brucellosis; Virulence factors; MultiplexPCR

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1.

INTRODUCTION

The World Health Organization (WHO) considers that brucellosis is one of the most common

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zoonotic infection (common diseases between humans and domestic animals) with a worldwide impact, contributing to significant health and economic problems (1-3). This infection transmitted to humans through consumption of unpasteurized dairy products or through direct contact with infected animals, placentas or aborted fetuses (1, 2). Although almost a century has

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gone by since its first description in the country, Iran has not been able to eradicate the disease

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and the population is at greater risk of acquiring brucellosis (3). Brucellosis is an old disease with minimal mortality. Yet human brucellosis remains the commonest zoonotic disease worldwide with more than 500,000 new cases annually, is associated with substantial residual disability, and is an important cause of travel-associated morbidity (4-6). The disease is caused by different species and four species namely Brucella melitensis (B. melitensis), Brucella suis (B. suis),

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Brucella abortus (B. abortus) and Brucella canis (B. canis) are the major causes of disease in human. Indeed, in Iran, which is considered as an endemic area of hygienic organization, B. melitensis is the most prevalent cause of this infection in human (7, 8). B. abortus is the most

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important zoonotic agent after B. melitensis. Although human infection with B. abortus may be

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mild, it can cause troublesome and intractable illness the occurrence of the acute, often incapacitating infection in man caused by B. melitensis (9, 10). This infection appears in the forms of the acute, sub-acute or chronic. In animals, Brucellosis often causes damage to the urinary-genital tract, but, in humans, it causes a severely debilitating and disabling illness, for weeks to months (2, 11). Additionally, brucellosis has major economic ramifications due to the time lost by patients from normal daily activities and losses in animal production (12, 13). Actually, Brucellosis has still remained as a public health threat around the world, especially in

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the Mediterranean region; consist of Iran, Turkey, the Arabian Peninsula, the Indian subcontinent, Mexico, and parts of central and south America (14, 15). The transmission of Brucella infection and its prevalence in a region depends upon several factors like food habits,

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milk processing methods and milk products, social customs, husbandry practices, climatic conditions, socioeconomic status, and environmental hygiene (16, 17). The pathogenesis of brucellosis, is mostly linked to the ability of Brucella to survive and replicate intracellular in host

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cells by expressing several cell envelope molecules contain integral Membrane-bound protein (MviN), Mannose-6-phosphateisomerase (ManA), Mannosyl-transferase (WbkA), Perosamine-

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synthetase (PerA), Outer membrane protein 19 (Omp19) that contribute to the control of the intracellular trafficking of the pathogen (18, 19). The other Brucella virulence factor is urease enzyme (Ure). Urease is an important virulence factor that plays a main role in the resistance of Brucella at low pH conditions, both in vivo and in vitro (20). An essential role in

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epidemiological studies, management of the outbreaks and control programs have the characterization of the bacterial critical enzymes and cell envelope virulence associated genes and also, the study of these virulence factors in Brucella spp. is important to evaluate

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pathogenicity of this bacteria (18, 21, 22). As regards, no article has documented the molecular characterization of the enzymes and cell envelope virulence associated genes of the circulating B.

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melitensis and B. abortus population in Iran, and in other hands, there are a few reports on the use of Multiplex-PCR assays for the detection of virulence factor of Brucella strains isolated from human and animals. So, the aim of the present study was to evaluate cell envelope associated virulence factor and urease enzyme among strains of B. melitensis and B. abortus isolated from clinical and non-clinical samples by Multiplex-PCR method in Iran. 2. Methods 4

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2.1. Bacterial isolation and examination In this cross-sectional study, 100 non-repetitive specimens suspected to brosellosis (include and animal cases during

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clinical and non-clinical samples) were obtained from human

December 2014 to April 2016. Clinical samples contained non-repetitive blood specimen obtained from patients suspected to brucellosis (sera-positive cases) referred to hospitals in

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Tehran, Arak and Hamedan. Non-clinical samples were recovered from animal blood and dairy products with symptoms of brucellosis that referred to veterinary clinics. Characteristics of

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human and animals participated in the current study are shown in Table 1. Samples were collected on the basis of clinical indication by standard procedure. Culture in isolation of Brucella spp. was carried out by standard technique, including cultured on Brucella agar (Oxoid, CM0169) with added Brucellas elective supplement (Oxoid, SR0083) and 5% of calf serum (Sigma, C8056), incubation at 37°C for 3 to7 days, use of Blood agar base and Nutrient agar; Selective

supplements

(Himedia-Accumax

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Brucella

India).

Brucella

isolates

were

phenotypically identified with colonial morphology, Gram stain, modified Ziehl-Nee1sen method and final identification with a series of conventional biochemical tests. Bacteriological

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diagnosis was confirmed by PCR technique. B. melitensis16M (ATCC 23456; NCTC 10094) and

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B. abortus544 (ATCC 23448; NCTC 10093) standard strains were used as controls. 2.2. DNA preparation

A loopful of colonies of each isolate on agar plate was and suspended in 300 µl of distilled water. After vortexing, the suspension was boiled for 8 min, and 150 µl of the supernatant was collected after centrifuging at 14,000 rpm for 10 min. The DNA concentration of the boiled extracts was determined by gel electrophoresis. We measure DNA purity and

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concentration using a NanoDrop™ spectrophotometer. The absorbance ratio at 260 nm and 280 nm (A260/280) is used to assess the purity of DNA and ratio of ~1.8 is considered as purity

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for extracted DNA. 2.3. Primers for detection of genus and species

Brucella Detection Primers B4/B5: These primers were located in the region within a gene

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coding for 31-kDa membrane protein specific to the genus Brucella were used for detection and diagnosis of Brucella species. Sequences of B4/B5 primers were show in Table 2 (23). In the

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study of Alamian et al., there was only one locus with the capacity of designing a proper primer to differentiate B. melitansis and B. abortus. So, PCR was performed to all Iranian isolates using specific primers that called UF1 and UR1. Sequences of forward and reverse primers for discrimination of B. melitansis and B. abortus were shown in the Table 2 (24).

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2.4. PCR assay for Identification of special species

PCR amplifications were performed in a final volume of 25 µL in PCR tubes. The reaction mixtures for detection of genus Brucella by B4/B5 primers and for differentiation of B.

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melitensis and B. abortus species by UF1/UR1 primers included 2.0 µL DNA template, 2.5 mM

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MgCl2, 0.4 µL (25 pmol) of each primer, 1.25 µL (1.25 U) of AmpliTaq DNA polymerase, 1 µL dNTPs (200 µM), 2.5 µL 10x PCR buffer (75 mM Tris-HCl, pH 9.0, 2 mM MgCl2, 50 mMKCl, 20 mM (NH4)2SO4, and sterile distilled water up to 25 µl volumes. These two reactions mixtures were prepared separately for performance of PCR. A PCR program for amplification of target sequences consisted of initial denaturation at 95°C for 4 min, 32 cycles of application with denaturation at 95°C for 30 seconds, annealing at 52°C for 30 seconds, extension at 72°C for 1 min and final extension of the incompletely synthesized DNA at 72°C for 7 min in the BioRad

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thermal cycler (MJ Mini, BioRad, USA). PCR program was equivalent into two steps of detection of Brucella genus and differentiation of B. melitensis/B. abortus species. The PCR products were analyzed in 2.0% agarose gels containing 1 µg /ml of power safe stain and

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subjected to electrophoresis in a 0.5X TBE buffer. Gels were visualized under UV light and documented using Bio-imaging Systems (VisiDoc-ItTM system). A molecular weight Ladder with

2.5. Multiplex-PCR for Identification of virulence genes

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100 bp increments (GeneRuler100 bp, plus DNA ladder) was used as a DNA standard.

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The target genes and primer sequences used in this study for detection of various virulence factors were demonstrated in Table 2. Multiplex PCR assays were conducted in a final volume of 25 µL in a BioRad thermal cycler (MJ Mini, BioRad, USA) comprising 12.5µl of GoTaq master mix (Promega, M2173) that contained Tris-HCl pH 8.5, (NH4) 2S04, 3 mM MgCl2,

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0.2% Tween 20, 0.4 mMdNTPs, 0.2 units/µL AmpliqonTaq DNA polymerase, Inert red dye and stabilizer, 0.4 µL of each primer (10 pmol, Syntheza), 2µl template DNA (0.5 µg) and sterile distilled water up to 25 µL. For Multiplex PCR technique, an additional set of primers were

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added to the reaction mixture. Thermal cycler was setup for initial denaturation at 95°C for 5 min, 35 cycles of application with 1 min denaturation at 95°C, 45 sec annealing at 58.5 °C and

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1min extension at 72°C. The final extension was at 72°C for 8 min. The Multi-stranded PCR product was analyzed by the electrophoresis technique on a 2% agarose gel for 1hour at 95 V and 30 mA, stained by power safe stain and was seen under UV transilluminator. Standard strains of B. melitensis 16M and B. abortus 544 were used as positive control strains and Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922 and Streptococcus pyogenes ATCC 19615 were used as negative control strains.

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2.6. Sequencing Analysis After amplification by PCR the virulence genes of positive control strains were sequenced (Bioneer Co., Korea, mediated by Takapouzist Co., Iran), and the data analysis were carried out

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by using the Chromas software. 2.7. Statistical analysis

The patient information (raw data), were entered in Excel 2010 (Microsoft Office, Microsoft,

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the Social Sciences (SPSS) software, ver. 16 (IBM).

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Washington D.C, USA) and Statistical analysis were performed using the Statistical Package for

3. RESULTS

Clinical samples were collected from several hospitals located across the provinces of Tehran, Arak and Hamadan in Iran, and the non-clinical samples were obtained from veterinary clinics in

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Tehran. The samples collected and used in the current study are given in Table 3. Out of the 100 suspected specimens obtained from clinical and non-clinical samples, using biochemical and microbiological techniques, 78 (78%) isolates were identified as Brucella genus. All 78 isolates

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were confirmed as Brucella spp. using PCR amplification method with specific primers. Agarose gel electrophoresis of PCR products for detection of Brucella genus is shown in Figure 1. The

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PCR amplification technique, with specific primers, further indicated that among the 78 Brucella spp. isolates, 57 (73%) and 21 (27%) strains were B. melitensis and B. abortus respectively (Table 3). The results of the PCR method for detection of B. melitensis and B. abortus species are shown in Figure 2.

Our results show that the Multiplex-PCR assay is a convenient and practical technique for simultaneous detection of multiple genes using gene-specific primers mixed in a single reaction.

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Multiplex PCR was performed to yield six bands in a single microtube, and the conditions were optimized with temperature, primer concentrations and extension time to avoid non-specific reaction. The sensitivity and specificity of the reaction were evaluated with respect to the

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reference strains. The Multiplex-PCR method was applied using specific primers for ure, wbkA, omp19, mviN, manA and perA genes that amplified in size 1282, 931, 550, 344, 271 and 716 bp respectively. The distribution of virulence-specific genes in the investigated clinical and animal

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brucellosis samples (n; 78, 78%) showed that 58 (74.4%), 70 (89.7%), 73 (93.6%), 74 (94.9%), 78 (100%) and 72 (92.3%) strains carried ure, wbkA, omp19, mviN, manA and perA genes

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respectively. Data obtained by performing Multiplex-PCR on all studied isolates are shown in Table 4. The properties of virulence genes and associated bands produced by Multiplex-PCR technique are presented in Figure 3.

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4. DISCUSSION

Brucellosis as a worldwide zoonosis disease remains an important economic and public health problem particular in developing countries such as Iran (25, 26). Brucella has a predilection for

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macrophages, dendritic cells (DCs) and trophoblasts (27). The bacteria can enter, survive, and replicate within these cells and cause disease (28). Brucella gains access to the host through

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inhalation, conjunctiva, skin abrasions and ingestion (29). The pathogens are later ingested by polymorphonuclear leukocytes and macrophages, and then transported to lymphoid tissue, such as lymph nodes, liver, spleen, mammary gland, bone marrow and reproductive tract (30, 31). According to Seleem et al. (2008), Brucella, as compared to other bacteria lack the classical virulence factors such as exotoxins, fimbria, capsules, plasmids, lysogenic phages, drug resistant forms, antigenic variation and endotoxic lipopolysaccharide (32). The pathogenicity of Brucella is due to its amazing ability to adapt to the environmental conditions encountered intracellularly 9

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(33, 34). Brucella has evolved to invade the host cell, evasion of the immune system, interfere with intracellular trafficking, resist respiratory burst, adapts to oxygen-limited conditions encountered inside macrophages, ability to enter and intracellular survival (32, 35). Brucella

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lacks classical virulence factors such as toxins or adherence, and its virulence seems ascribed to type 4 secretion system, different enzymes, lipopolysaccharide and the peculiar properties of the cell envelope (34, 36). Cell envelope proteins are one of important virulence factors of Brucella

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species that contributes to entry and initial survival of bacteria in macrophages and thereby increased the potential virulence of this pathogen (37). Some recent studies showed that the

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function of certain cell envelope associated genes was environmental stress adaptation, intracellular-modulatory activity and ability to survive (23). Thus, the presence of such genes in the Brucella genome will confirm that this bacterium is a pathogen. According to Cloeckaert et al. study (2003), cell envelope factors are the most significant virulence factors for Brucella

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which allowed for survival inside macrophages and other cells of the reticuloendothelial system (38). Interaction between pathogens and hosts initiates a dynamic cascade of signals that lead to the change of gene expression patterns in the cell envelope proteins, which results in either

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colonization or elimination of pathogens in hosts (18, 39, 40). To our knowledge, up to now, no article has documented the molecular characterization of cell envelope virulence associated

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genes in the circulating B. melitensis and B. abortus strains in Iran. According to the study conducted by Awwad et al. (2015), among 80 non-clinical isolates of Brucella, all of them possess the omp19 gene and also more than 95% of them have manA and perA genes by Multiplex-PCR (18). This data was very close to our results so that we show 87.1%, 93.5% and 100% of non-clinical isolates have omp19, perA and manA, respectively. In the research carried out by Razzaq and colleagues (2014), the wbkA and mviN genes were found in 100% of clinical

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Brucella species. isolated from human blood samples and also manA was presented in 87.5% of these samples. In other hand, manB and omp31 markers were detected in all studied strains, while the omp25 gene was founded only in 50% of isolates (23). In a study conducted by Naseri

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et al. (2016), the frequencies of wbkA and manA genes among 57 B.melitensis strains isolated from human blood samples were reported to be 100% in contrast to the results of another study by Khosravi et al. (2006) (41, 42). These data were similar to the results of our study because we

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also reported the high prevalence of envelop virulence genes wbkA, mviN and manA in the investigated strains. On the other hand, one of the essential virulence factors for pathogenicity of

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Brucella spp. is urease enzyme encoded by two separate urease gene clusters (ure1 and ure2 genes) (20). Ureases are multi-subunit metallo-enzymes that hydrolyze urea to form carbonic acid and two molecules of ammonia; the ammonia molecules protonate to form ammonium causing the pH to increase. The degradation of urea provides ammonium for incorporation into

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intracellular metabolites and facilitates survival in acidic environments (43, 44). Our results confirm that the prevalence of ure gene in clinical samples was 78.7%. The study by Derakhshandeh et al. (2013) showed that the frequency of ure gene was 88% among clinical

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specimens isolated from aborted fetuses that agree (45). These results are in disagreement with the present study. Other studies show most Brucella isolates exhibit potent urease activity that

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has been hypothesized to play a role in the pathogenesis of disease (46). Thus, one could argue that the outer membrane contains various cell envelope proteins along with other virulence factors such as urease enzyme play an essential role in the process of pathogenesis of bacteria (47). For further assessment of these genes, biogenesis, structure, mechanism, activity and their roles in pathogenesis pathway of pathogenic strains more researches will be necessary. Moreover, we could apply the Multiplex-PCR method for spontaneous detection of several

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virulence genes in pathogenic species of Brucella and obtained appropriate results that indicate to the efficiency of Multiplex PCR for investigation and verification of Brucella virulent strains

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and accurate/fast detection of these strains. Although brucellosis has been, or is close to being, eradicated from a number of developed countries, it continues to be a major public and animal health problem in many regions of the

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world. The results of the present study shows that most B. melitensis and B. abortus isolates from Iran have the most virulence factor genes (wbkA, omp19, mviN, manA and perA) in their genome,

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but ure gene more frequently existed in B. melitensis strains that because of the critical role of urease in pathogenesis it has been hypothesized to B. melitensis is more virulent than B. abortus. So, each step in the Brucella infection process can be mediated by a number of virulence factors and each strain may have a unique combination of these factors that affected the rate of bacterial pathogenesis. This characterization has a pivotal role in epidemiological studies, management of

Acknowledgement:

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the outbreaks and implementing controls and preventive measures.

The authors are greatly thankful to Saadat Pirouzi for help with the English language version of

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this paper. Also, authors would like to thank the director and principal of Iran university of

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medical sciences for their constant encouragement and support for the current study. Conflict of interest:

The authors declare that there is no conflict of interests in the current study. Funding:

No Funding.

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VirB12 recombinant protein is a potential marker for serodiagnosis of human brucellosis. Ann Clin Microbiol Antimicrob. 2017;16:8-13. 34. Mirnejad R, Piranfar V, Mortazavi SM, Kachuei R. A duplex PCR for rapid and simultaneous detection of Brucella spp. in human blood samples. Asian Pac J Trop Med.

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35. Lopez-Goni I, Guzman-Verri C, Manterola L, Sola-Landa A, Moriyon I, Moreno E.

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Regulation of Brucella virulence by the two-component system BvrR/BvrS. Vet Microbiol. 2002;90:329-39.

36. Guzmán-Verri C, Manterola L, Sola-Landa A, Parra A, Cloeckaert A, Garin J, et al. The two-component system BvrR/BvrS essential for Brucella abortus virulence regulates the expression of outer membrane proteins with counterparts in members of the Rhizobiaceae. Proc Natl Acad Sci. 2002;99:12375-80. 37. Brambila-Tapia AJL, Armenta-Medina D, Rivera-Gomez N, Perez-Rueda E. Main Functions and Taxonomic Distribution of Virulence Genes in Brucella melitensis 16 M. PloS One. 2014;9:1-10. 15

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38. Cloeckaert A, Grayon M, Grépinet O, Boumedine KS. Classification of Brucella strains isolated from marine mammals by infrequent restriction site-PCR and development of specific PCR identification tests. Microbes Infect. 2003;5:593-602. 39. N Xavier M, A Paixao T, B den Hartigh A, M Tsolis R, L Santos R. Pathogenesis of

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common virulence-associated genes among Brucella Melitensis isolates from human blood cultures in Hamadan Province, West of Iran. Iran J Med Sci. 2016;41:422-8.

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42. Khosravi AD, Abassi E, Alavi SM. Isolation of Brucella melitensis and Brucella abortus from brucellosis patients by conventional culture method and polymerase chain reaction technique. Pak J Med Sci. 2006;22:396-400.

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44. Wray LV, Ferson AE, Fisher SH. Expression of the Bacillus subtilis ureABC operon is controlled by multiple regulatory factors including CodY, GlnR, TnrA, and Spo0H. J Bacteriol. 1997;179:5494-501.

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46. Smith LD, Ficht TA. Pathogenesis of brucella. Crit Rev Microbiol. 1990;17:209-30. 47. He Y. Analyses of Brucella pathogenesis, host immunity, and vaccine targets using systems biology and bioinformatics. Front Cell Infect Microbiol. 2012;2:1-17.

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Table Legends:

Table 1. Characteristics of the patients who participated in this study

Table 3. Descriptive statistics for the bacterial isolation

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Table 2. Sequences of all primers used in this study

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Table 4. Prevalence of the studied virulence genes in the Brucella species by Multiplex PCR

Figure Legends:

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Figure 1. PCR products for detection of genus Brucella (amplified by B4, B5 primer pair). Lane L: DNA Ladder 50 bp; Lane 1: positive control (B. abortus544 [ATCC 23448]); Lane 2-5: strains of genus Brucella isolated from clinical and non-clinical samples (band 223 bp); Lane 6: Pseudomonas aeruginosa ATCC 27853.

Figure 2. PCR products for detection of B. melitensis and B. abortus species (amplified by UF1,

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UF2 primer pair). Lane L: DNA Ladder 100 bp; Lane 1: Escherichia coli ATCC 25922; Lane 2-9: strains of B. melitensis and B. abortus species isolated from clinical and non-clinical samples (band 99 bp); Lane 10: positive control (B. melitensis16M [ATCC 23456]); Lane 11: Pseudomonas aeruginosa ATCC 27853.

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Figure 3. Multiplex-PCR amplification of the ure, wbkA, omp19, mviN, manA and perA genes in B. melitensis and B. abortus isolates. Lane 1: DNA Ladder 100 bp; Lane 2: positive control

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Lane 3-12: strains isolated from clinical and animal samples. Lane 13: Pseudomonas aeruginosa ATCC 27853. Lane 14: Escherichia coli ATCC 25922. [band 1282 bp: ure, band 931 bp: wbkA, band 550 bp: omp19, band 344: mviN, band 271 bp: manA, band 716 bp: perA].

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Table 1 Characteristics of the patients who participated in this study

Sex

Descriptive

Index

Index per

No. (%)

Location

T

A

H

Male

39 (81.25%)

19

9

11

Female

8 (18.75%)

4

1

3

-

Age (mean± SD)

35±18 years

Under diploma

31(68%)

17

6

8

11(23.4%)

6

3

2

5 (10.6%)

0

1

4

Master’s degree

0

0

0

0

Doctorate

0

0

0

0

28 (59.5%)

11

8

9

19 (40.5%)

12

2

5

Cattle

11 (36.6%)

7

2

2

Sheep

16 (53.3%)

10

4

2

Goat

4 (13.4%)

3

0

1

Diploma

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Education

Bachelor’s degree

Living

Rural

Animals

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Urban Non-clinical

Descriptive

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clinical

Characteristics

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Isolates

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T=Tehran, A= Arak, H=Hamadan

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Table 2 Sequences of all primers used in this study Primers sequence

Amplified product Size (bp)

Ref.

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Primer name

Primer sequences used for detection of Brucella genus B4 (Forward) B5 (Revers)

F: 5'-TGGCTCGGTTGCCAATATCAA-3' R: 5'-CGCGCTTGCCTTTCAGGTCTG-3'

223

(23)

F: 5'-GGCTATCGGCTGGGAAAGG-3' R: 5'-CCTTCCGAAGAAAATACCCCT-3'

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UF1 (Forward) UR1 (Revers)

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Primer sequences used for detection of B. abortus and B. melitensis species 99

(24)

Primer sequences used for detection of virulence genes in Brucella F: 5'-TGATGGGAATTTCAAAAGCA-3' R: 5'-GTTTCCGGGTCAGATCAGC-3'

550

(18)

wbkA

F: 5'-AATGACTTCCGCTGCCATAG-3' R: 5'-ATGAGCGAGGACATGAGCTT-3'

931

(18)

manA

F: 5'-TCGATCCAGAAACCCAGTTC-3' R: 5'-CATACACCACGATCCACTGC-3'

271

(23)

mviN

F: 5'-GCAGATCAACCTGCTCATCA-3' R: 5'-GGCCATAGATCGCCAGAATA-3'

344

(23)

F: 5'-GCTTGCCCTTGAATTCCTTTGTGG-3' R: 5'-ATCTGCGAATTTGCCGGACTCTAT-3'

1282

(45)

716

(18)

perA

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ure

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omp19

F: 5'-GGAACGGTGGCACTACATCT-3' R: 5'-GGCTCTCTGTGTTCCGAGTT-3'

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No. (%)

samples

Brucella spp. isolates

strains

B. melitensis

No. (%)

Blood

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Clinical

B. abortus

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Samples

63 (63%)

47 (74.6%)

Blood

23 (23%)

20 (87%)

16 (80%)

4 (20%)

Dairy Products

14 (14%)

11 (78.6%)

8 (72.7%)

3 (27.3%)

Total

100

78 (78%)

57 (73%)

21 (27%)

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Non-Clinical

33 (70.2%)

No. (%)

14 (29.8%)

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Table 4 Prevalence of the studied virulence genes in the Brucella species by Multiplex PCR Total

Target

No.

sample

strains

(%) wbkA

omp19

mviN

manA

perA

33

30

31

33

33

33

30

(70.2%)

(90.9%)

(93.9%)

(100%)

(100%)

(100%)

(90.1%)

63

B. abortus

14

7

12

13

14

14

13

(63%)

(29.8%)

(50%)

(85.7%)

(92.9%)

(100%)

(100%)

(92.9%)

total

47

37

43

46

47

47

43

(78.7%)

(91.5%)

(97.9%)

(100%)

(100%)

(91.5%)

18

21

21

20

24

24

(75%)

(87.5%)

(87.5)

(83.3%)

(100%)

(100%)

3

6

6

7

7

5

(22.6%)

(42.9%)

(85.7%)

(85.7%)

(100%)

(100%)

(71.4%)

31

21

27

27

27

31

29

(100%)

(74.2%)

(87.1%)

(87.1%)

(87.1%)

(100%)

(93.5%)

78

58

70

73

74

78

72

(78%)

(74.4%)

(89.7%)

(93.6%)

(94.9%)

(100%)

(92.3%)

(100%)

B. melitensis

24 (77.4%)

Non37

B. abortus

(37%)

100

-

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Total

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total

7

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Clinical

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Clinical

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ure

s B. melitensis

No. of virulence genes in studied strains of Brucella (%)

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Samples

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223 bp

FIGURE 1. PCR products for detection of genus Brucella (amplified by B4, B5 primer pair). Lane L: DNA Ladder 50 bp; Lane 1: positive control (B. abortus544 [ATCC 23448]); Lane 2-5: strains of genus Brucella isolated from clinical and non-clinical samples (band 223 bp); Lane 6: Pseudomonas aeruginosa

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ATCC 27853.

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99 bp

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100 bp

FIGURE 2. PCR products for detection of B. melitensis and B. abortus species (amplified by UF1, UF2 primer pair). Lane L: DNA Ladder 100 bp; Lane 1: Escherichia coli ATCC 25922; Lane 2-9: strains of B. melitensis and B. abortus species isolated from clinical and non-clinical samples (band 99 bp); Lane 10: positive control (B. melitensis16M [ATCC 23456]); Lane 11: Pseudomonas aeruginosa ATCC

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27853.

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1282 bp 931 bp

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716 bp 550 bp 344 bp

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271 bp

FIGURE 3. Multiplex-PCR amplification of the ure, wbkA, omp19, mviN, manA and perA genes in B. melitensis and B. abortus isolates. Lane 1: DNA Ladder 100 bp; Lane 2: positive control; Lane 3-12:

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strains isolated from clinical and animal samples; Lane 13: Pseudomonas aeruginosa ATCC 27853; Lane 14: Escherichia coli ATCC 25922. [Band 1282 bp: ure, band 931 bp: wbkA, band 550 bp: omp19,

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band 344: mviN, band 271 bp: manA, band 716 bp: perA].

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Highlights •

Brucellosis (as a Life-threatening disease) is a dangerous infection that causes many

•

Brucellosis is one of the main health problems in Iran (particular in rural areas) and neighboring countries (especially in Middle-East).

The most of Brucella strains isolated from different regions of Iran possess the most

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•

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complications in patients and endangers patient's life.

virulence factor genes (ure, wbkA, omp19, mviN, manA and perA) in their genome. The ure gene more frequently existed in B. melitensis strains that because of the critical

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•

role of urease in pathogenesis it has been hypothesized to B. melitensis is more virulent than B. abortus. •

Each step of Brucella infection can be mediated by several virulence factors and each

pathogenesis.

This characterization has a pivotal role in epidemiological studies, management of the

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outbreaks and implementing controls and preventive measures.

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•

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strain may have a unique combination of these factors that affected the rate of bacterial

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AUTHORS’ CONTRIBUTION Reza Mirnejad contributed to the conception and design of the work; the acquisition, analysis, and interpretation of data for the work. Mansour Sedighi contributed to data collection and

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interpretation of data for the work. Shayan Mostafaei contributed in data analysis, Drafting the work and revising it critically for important intellectual content. Mansour Sedighi and Faramarz Masjedian Jazi contributed in the revising the draft and agreement to be accountable for all

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aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Reza Mirnejad and Faramarz Masjedian

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Jazi contributed in revising the article and final approval of the version to be published.