Molecular typing of Brucella species isolates from Human and livestock bloods in Isfahan province
Ebtehaj Pishva1, Rasoul Salehi2, Akbar Hoseini3, Ali Kargar3, Fatemeh Emani Taba3, Maryam Hajiyan3, Reza Fadaei1, Javad Ramezanpour4
1 Department of Microbiology and Virology, Isfahan University of Medical Sciences, Isfahan, Iran
2 Department of Genetics and Molecular Biology, Medical School, Isfahan University of Medical Sciences, Isfahan, Iran
3 Center of Veterinary and Clinic Network, Isfahan University of Medical Sciences, Isfahan, Iran
4 Department of Public Health, Isfahan University of Medical Sciences, Isfahan, Iran
|Date of Submission||22-Dec-2013|
|Date of Acceptance||12-May-2014|
|Date of Web Publication||29-May-2015|
Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan
Source of Support: None, Conflict of Interest: None
Background: Human brucellosis is caused by infection with certain species of the genus Brucella and is characterized by bacterial persistence and inflammation of many host tissues. Handling all live Brucella involves risk of laboratory infection and very strict biosafety rules must be observed. In order to avoid these disadvantages, method based on the PCR-RFLP shows excellent typeability, reproducibility, stability, and epidemiological concordance. The omp2 locus contains two gene copies (named omp2a and omp2b) coding for porin proteins and has been found particularly useful for molecular typing and identification of Brucella at the species, biovar, or strain level. This study is designed to evaluate the molecular epidemiology of Brucella spp from human and livestock in Isfahan province, central region of Iran in order to use the findings in efficient disease prevention programs.
Materials and Methods: One hundred ninety blood samples were collected from human and cattle with active brucellosis and 40 aborted ewes fetuses were collected and genotyped using PCR-RFLP technique, DNA polymorphisms such as the restriction patterns of the PCR-amplified omp2a and omp2b genes.
Results: The molecular characterization performed to assess the species and the biovar of the Brucella strains. Analysis of the 230 isolates examined in this study generated three unique RFLP profiles. One of the profiles was the most common being present in 134/180.
Conclusion: Our findings confirm abundance of B. melitensis, particularly biovar 1 in human and sheep are identical but B. abortus biovar 3 as the etiological agent of cattle brucellosis most frequently isolated in the Isfahan area.
Keywords: Brucella, brucellosis, molecular typing, omp2, PCR-RFLP
|How to cite this article:|
Pishva E, Salehi R, Hoseini A, Kargar A, Taba FE, Hajiyan M, Fadaei R, Ramezanpour J. Molecular typing of Brucella species isolates from Human and livestock bloods in Isfahan province. Adv Biomed Res 2015;4:104
|How to cite this URL:|
Pishva E, Salehi R, Hoseini A, Kargar A, Taba FE, Hajiyan M, Fadaei R, Ramezanpour J. Molecular typing of Brucella species isolates from Human and livestock bloods in Isfahan province. Adv Biomed Res [serial online] 2015 [cited 2023 Jun 6];4:104. Available from: https://www.advbiores.net/text.asp?2015/4/1/104/157798
| Introduction|| |
Brucellosis, a typical zoonotic disease, systematic infection transmitted from animals (wild or domestic) to human, remains a worldwide veterinary and medical problem contributing to health and economic situation of affected regions.  The source of naturally acquired brucellosis in humans is almost always to be found in the animal reservoirs although very few cases of human to human transmission have also been reported. ,
Brucellosis is present throughout the five continents and it is still an uncontrolled serious public health problem in many developing countries. , It is endemic in sheep and goats in most countries of the Mediterranean.  Distinction between the Brucella species and biovars in the past was mainly based on phenotypic characteristics.  Traditional methods of identifying Brucella to the species level is time consuming, subjective, and can pose a hazard to laboratory personnel in the absence of suitable biocontainment facilities. ,
The omp2 locus contains two gene copies (named omp2a and omp2b) coding for porin proteins and has been found particularly useful for molecular typing and identification of Brucella at the species, biovar, or strain level. 
Today, with advancement of molecular techniques, several molecular typing methods to find DNA polymorphism are used, which are capable to identify Brucella biovars and species. , polymerase chain reaction-fragment length polymorphism (PCR-RFLP) is a common approach for typing of Brucella spp., providing a good tool for taxonomic, epidemiological, evolutionary and diagnostic studies. ,, The method has especially been utilized in studies of various outer membrane protein (omp) genes.  Several Brucella omp genes were considered as potential targets for typing schemes, however, the Omp2 locus [Omp2a and Omp2b) shows greatest polymorphism among the Brucellae spp. , No data, however, are available about the molecular epidemiological characterization of Brucella isolates from humans and livestock bloods. In this study, the genetic profiles of B. melitensis and B. abortus isolates from humans and animals were analyzed and compared by PCR-RFLP of omp2a and omp2b genes; a specific marker was detected grouping the human and animals Brucella spp.  PCR-RFLP of the omp2 locus has good reproducibility and has been useful for differentiation of Brucella species, even though it is somewhat limited by the lack of natural sequence differences at the biovar level.  This study was designed to evaluate the molecular epidemiology of Brucella spp from human and livestock in the Isfahan area and applying the findings to planning for disease control in this province.
| MATERIALs AND METHODS|| |
We collected 150 human blood samples from outpatients with active brucellosis. These patients were seen in health centers in Isfahan province during March 2009 to April 2010. STA tests for all the patients were positive (titers of; 100 IU/mL) at the beginning of the disease. We also collected bovine blood samples from 40 cattle. The blood collection from these cattle was done to determine bovine seropositivity . 5 ml blood was taken from patients and bovine blood samples were transferred to laboratory and were kept at −20°C.
Extraction of genomic DNA
DNA was extracted from the blood specimens using a commercial purification system (Wizard Genomic DNA Purification Kit, Promega, Madison, WI) according to the manufacturer's instructions for DNA purification from blood. Final pellets were resuspended in 50 μL of TE (10 mMTris, 1 mM EDTA, pH 7.2). ,
Samples from abomasum content and liver of the ewes' aborted fetuses were inoculated onto blood agar plates containing 7% defibrinated sheep blood with and without Brucella supplement (Oxoid, SR083A). The cultures were incubated at 37°C under an atmosphere with 10% CO2. Brucella were identified by morphological, cultural and biochemical characteristics such as oxidase, urease, CO2 requirement, H2S production, growth in the presence of thionin and basic fuchsin (20 μg/ml). The strains were bio typed by agglutination with mono specific A and M antiserum.
DNA preparation for Brucella genomic DNA extraction, Brucella sample strains were cultured for 24 h at 37°C on tryptic soy agar-yeast extract slopes and harvested, in 3 ml of sterile distilled water, by centrifugation at 2,000 × g for 10 min. The pellet was suspended in 567 μl of TE/sodium buffer (50 mM Tris, 50 mM EDTA, and 100 mM NaCl [pH 8.0]). Then, 30 μl of 10% (wt/vol) sodium dodecyl sulfate (SDS) solution and 3 μl of 2% (wt/vol) proteinase K solution were added, and the mixture was kept at 37°C for 1 h. The lysed cell suspension was extracted twice with phenol-chloroform, and nucleic acids were precipitated by gently mixing the aqueous phase with 2 volumes of cold ethanol. The precipitate was dissolved in 100 μl of TE (10 mM Tris, 1 mM EDTA [pH 8.0]).
The amount of DNA was measured by electrophoresis of an aliquot of each sample through 0.8% agarose gels and was compared with standard DNA solutions  . The extracted DNA was then used for PCR amplification of part of omp2a gene.
The Brucella omp2a gene was used as target DNA for PCR amplification.  Specific oligonucleotides (omp2aF 5'-CCTTCAGCCAAATCAGAATG-3', omp2aR 5'-GGTCAGCATAAAAAGCAAGC-3') were used for amplification of this gene locus. ,
The reaction mixture contained 5 μL of 10x PCR buffer, 10 pMol of the primer mix, 0.2 μMol of dNTP mix, 1.5 mM MgCl 2 , 1 unit of Taq DNA polymerase, 100 pg of sample DNA in a volume of 50 μL. No template negative controls were routinely processed to monitor contamination with Brucella DNA and positive controls were included to monitor amplification success. Amplifications were initiated by denaturing the sample for 3 min at 95°C, followed by 30 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s. After the last cycle, samples were incubated for an additional 5 min at 72°C before storage at 4°C. 
Five microliters of PCR-amplified omp2a gene was digested by the restriction enzyme PstI (Promega Corporation, Madison, WI), at 37°C for 3 h in a 20 μL reaction volume using manufacturer's recommended buffer. The mixtures were then electrophoresed on a 2% agarose gel containing DNA green viewers. 
Statistical analysis was conducted to determine how many samples were positive for Brucell spp biovar, as well as those positive for two Brucella spp. Perspective analyses were performed and data rounded numerical values (percentage) were documented.
| Results|| |
The molecular characterization performed to assess the species and the biovar of the Brucella strains. Analysis of the 230 isolated examined in this study generated three unique profiles. One of the profiles was the most common being present in 134/180. ,, This profile was predominantly associated with 74% of the isolate but was also less frequently seen in cattle [Table 1].
|Table 1: RFLP patterns detected when PCR amplified omp2a gene of the human and livestock Brucella strains digested by Pst1 enzyme |
Click here to view
According to our findings, since 62% of human and sheep isolates were identified as Brucella melitensis biovar 1 [Figure 1] pattern 3], 35% B. melitensis biovar 1,2 and B. abortus biovar 3, 5, 6 Figure and [Table 1] pattern 1, 5% B. abortus biovar 1, 2, 4 and B. melitensis biovar 1, 2, 3 Figure and [Table 1] pattern 2. B. melitensis (biovars 1 and 3) is the predominant species, associated with sporadic cases and outbreak in humans and sheep. In cattle the biovar of the Brucella strains indicated the presence of 20% isolates of B. abortus biovar 1, 2, 4 and B. melitensis biovar 1, 2, 3 Figure and [Table 1] pattern 2, 70% B. abortus biovar 3, 5, 6
|Figure 1: Restriction patterns of PCR amplified omp2a genes of the human and livestock Brucella strains digested by Pst1 enzyme|
Click here to view
B. melitensis biovar 1, 2 Figure and [Table 1] pattern 1, 10% B. melitensis biovar 1 [[Figure 1] pattern 3]. In particular, B. abortus biovar 3, 5, 6 was significantly associated with cattle (70% versus 5% in sheep and goat), while B. melitensis biovar 1 was significantly associated with human and sheep and goat (62% versus 10% in cattle).
| Discussion|| |
Human brucellosis is caused by infection with certain species of the genus Brucella and is characterized by bacterial persistence and inflammation of many host tissues.  Neutrophils are one of the predominant cell types present in the infiltrate of these inflamed tissues, and due to their potential effect on the inflammatory response and tissue damage, direct activation of neutrophils by Brucella abortus might contribute to the pathology associated with human brucellosis.  B. abortus expresses outer membrane lipoproteins (Omp) with inflammatory properties on a variety of cell types. 
B. melitensis is the most important zoonotic agent, followed by B. abortus and B. suis. The occurrence of brucellosis in human is directly related to the status of animal brucellosis in a particular geographical region. , Hence, defining a case as brucellosis with the causative species will give more clues about prevalence, treatment, and control measures rather than merely diagnosing as brucellosis.  Brucella species vary from country to country, however the most common isolated species are B. melitensis and B. abortus., Although B. abortus is not usually associated with human brucellosis, it is the most common cause of the infection in general. Its clinical picture is less severe than the one seen in infections caused by B. melitensis.  B. melitensis is the most frequently reported cause of human brucellosis and it is commonly isolated from the patients.  As the most virulent type, B. melitensis is related with severe, acute diseases. In some regions it is reported as endemic. In Iran, brucellosis was first recognized in 1949  and is now endemic throughout the country. The annual reported incidence of human diseases is 26551 cases in 2005. The prevalence of brucellosis among sheep/goats was 10.2% and among cows was 17.5% in the 1990s. The long-term serological studies have indicated that 5% of sheep and 0.8% of cattle was infected with brucellosis.  In our country, concordantly, B. melitensis is known to be the main cause for human brucellosis. , Due to differences in the pathogenicity of Brucella species and biovars, with a view to epidemiology of brucellosis, recognition of Brucella biovars is important and hence typing of various strains is the main task of control centers of brucellosis and must be performed continuously. ,, In this study, the genetic profiles of B. melitensis and B. abortus isolates from humans and animals were analyzed by DNA polymorphisms at the omp2 locus. The omp2 locus contains two gene copies (named omp2a and omp2b) coding for porin proteins and has been found particularly useful for molecular typing and identification of Brucella at the species, biovar, or strain level.  On the basis of restriction patterns (from 13 restriction enzymes) of the PCR-amplified omp2a genes Brucella strains could be classified into different groups [Figure 1].  How ever PCR-RFLP of omp2a and omp2b was not able to accurately identify our isolates, which were identified as B. melitensis biovar 1 or 3 and B. abortus biovars [[Figure 1] Patterns 1, 2]. , In the province of Isfahan, brucellosis is an endemic disease and the main sources of brucellosis in animals and humans are infected sheep and cattle. B. melitensis biovars 1 is the predominant species, associated with sporadic cases and outbreak in humans. Isolates of B. abortus, primarily biovars 1, 2, 3 are also associated with sporadic human brucellosis. [ 23],
| Conclusion|| |
Our findings confirm abundance of B. melitensis, particular biovar 1 in human and sheep are identical but B. abortus biovar 3 as the etiological agent of cattle brucellosis most frequently isolated in Isfahan area and suggest, therefore, that sheep and goat populations are the principal cause of human brucellosis.
| Acknowledgments|| |
This work was financially supported by Isfahan University of Medical Sciences, Deputy for Research (grant number 185234). We also would like to thank members of Genetics and Molecular Biology as well as Bacteriology and Virology Departments of Medical School of Isfahan University of Medical Sciences.
| References|| |
Pappas G, Papadimitriou P, Akritidis N, Christou L, Tsianos EV. The new global map of human brucellosis. Lancet Infect Dis 2006;6:91-9.
Godfroid J, Scholz HC, Barbier T, Nicolas C, Wattiau P, Fretin D, et al
. Brucellosis at the animal/ecosystem/human interface at the beginning of the 21 st
century. Prev Vet Med 2011;102:118-31.
Vizcaino N, Kittelberger R, Cloeckaert A, Marin CM, Fernandez-Lago L. Minor nucleotide substitutions in the omp31 gene of Brucella ovis
result in antigenic differences in the major outer membrane protein that it encodes compared to those of the other Brucella
species. Infect Immun 2001;69:7020-8.
Cloeckaert A, Vizcaino N, Paquet JY, Bowden RA, Elzer PH. Major outer membrane proteins of Brucella spp
.: Past, present and future. Vet Microbiol 2002;90:229-47.
Carvalho Neta AV, Mol JP, Xavier MN, Paixão TA, Lage AP, Santos RL. Pathogenesis of bovine brucellosis. Vet J 2010;184:146-55.
Salehi M, Pishva E, Salehi R, Rahmani R. Isolation of Brucella
abortus using PCR-RFLP analysis. Iran J Pub Health 2006;35:22-27.
De Massis F, Di Girolamo A, Petrini A, Pizzigallo E, Giovannini A. Correlation between animal and human brucellosis in Italy during the period 1997-2002. Clin Microbiol Infect 2005;11:632-6.
Corbel MJ. Identification of dye-sensitive strains of Brucellamelitensis
. J Clin Microbiol 1991;29:1066-8.
Al Dahouk S, Scholz HC, Tomaso H, Bahn P, Göllner C, Karges W, et al
. Differential phenotyping of Brucella
species using a newly developed semi-automated metabolic system. BMC Microbiol 2010;10:269.
Zerva L, Bourantas K, Mitka S, Kansouzidou A, Legakis NJ. Serum is the preferred clinical specimen for diagnosis of human brucellosis by PCR. J Clin Microbiol 2001;39:1661-4.
Lucero NE, Ayala SM, Escobar GI, Grayon M, Jacques I. A new variant of Brucellamelitensis
. Clin Microbiol Infect 2006;(12):593-6.
Cloeckaert A, Vizcaino N, Paquet JY, Bowden RA, Elzer PH. Major outer membrane proteins of Brucella spp
.: Past, present and future. Vet Microbiol 2002;90:229-47.
Cloeckaert A, Verger JM, Grayon M, Paquet JY, Garin-Bastuji B, Foster G, et al
. Classification of Brucella spp
. isolated from marine mammals by DNA polymorphism at the omp2 locus. Microbes Infect 2001;3:729-38.
Al Dahouk S, Tomaso H, Prenger-Berninghoff E, Splettstoesser WD, Scholz HC, Neubauer H. Identification of Brucella
species and biotypes using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Crit Rev Microbiol 2005;31:191-6.
Nagalingam M, Shome R, Balamurugan V, Shome BR, NarayanaRao K, Vivekananda, et al
. Molecular typing of Brucella
species isolates from livestock and human. Trop Anim Health Prod 2012;44:5-9.
Leal-Klevezas DS, Martinez-Vazquez IO, Lopez-Merino A, Martinez- Soriano JP. Single-step PCR for detection of Brucella
spp. From blood and milk of infected animals.J Clin Microbiol 1995;33:3087-95.
Marianelli C, Ciuchini F, Tarantino M, Pasquali P, Adone R. Molecular characterization of the rpoB gene in Brucella
species: New potential molecular markers for genotyping. Microbes Infect 2006;8:860-5.
Cloeckaert A, Grayon M, Grepinet O. Identiﬁcation of Brucellamelitensis
vaccine strain Rev.1 by PCR-RFLP based on a mutation in the rpsL gene. Vaccine 2002;20:2546-50.
Baddour MM, Alkhalifa DH. Evaluation of three polymerase chain reaction techniques for detection of Brucella
DNA in peripheral human blood. Can J Microbiol 2008;54:352-7.
Pishva E, Salehi M, Gharavi M. Relationship between Brucella
immunocomplex and glomerulopathies. J Clin Infect Dis 2008;5:127-32.
Whatmore AM, Murphy TJ, Shankster S, Young E, Cutler SJ, MacMillan AP. Use of amplified fragment length polymorphism to identify and type Brucella
isolates of medical and veterinary interest. J Clin Microbiol 2005;43:761-9.
Ancora M, De Santis P, Di Giannatale E, Luciani M, Alessiani A. Molecular typing of Brucella
field strains isolated in Italy. Vet Ital 2005;41:51-5.
Kutlu G, Ertem GT, Coskun O, Ergun U, Gomceli YB, Tulek N, et al
: A cause of peripheral neuropathy. Eur Neurol 2009;61:33-8.
Cloeckaert A, Verger JM, Grayon M, Grepinet O. Restriction site polymorphism of the genes encoding the major 25 kDa and 36 kDa outer membrane proteins of Brucella
. Microbiology 1995;141:2111-21.