Original article
Determination of investigation of the link between human and animal Brucella isolates in Iran using multiple-locus variable number tandem repeat method comprising 16 loci (MLVA-16)
Shiva Mirkalantari
, Faramarz Masjedian
, Amraee Fateme
Corresponding author
Corresponding author
Iran University of Medical Sciences, Faculty of Medicine, Microbiology Department, Tehran, Iran
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The columns represent the number of strains, geographical origin and bacterial sources.</p>" ] ] ] "textoCompleto" => "<span class="elsevierStyleSections"><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0040">Introduction</span><p id="par0005" class="elsevierStylePara elsevierViewall">Brucellosis is a zoonotic bacterial disease caused by the genus Brucella, which is transmitted through direct or indirect contact to animals.<a class="elsevierStyleCrossRef" href="#bib0005"><span class="elsevierStyleSup">1</span></a> Based on differences in pathogenicity, phenotypic characteristics and hosts, the genus Brucella is classified into 11 species.<a class="elsevierStyleCrossRefs" href="#bib0010"><span class="elsevierStyleSup">2,3</span></a> Four species, including Brucella abortus, Brucella melitensis, <span class="elsevierStyleItalic">Brucella canis</span> and <span class="elsevierStyleItalic">Brucella suis</span> are known to infect humans. Enormous economic losses and public health problems results from abortion, infertility in livestock, weak offspring, decreased milk production, and morbidity in humans. Although brucellosis has been eradicated in the USA, Canada, North Europe, and Australia, this infection is still highly prevalent in central Asia, Middle East, Mediterranean region, Africa, and Latin America.<a class="elsevierStyleCrossRef" href="#bib0020"><span class="elsevierStyleSup">4</span></a> Iran is an endemic area for brucellosis and there is a major risk of Brucella transmission from eastern and western neighboring countries such as Iraq, Pakistan, and Afghanistan due to lack of high quality veterinary services for controlling animal diseases.<a class="elsevierStyleCrossRefs" href="#bib0025"><span class="elsevierStyleSup">5,6</span></a> Although, 500,000 human cases of brucellosis are annually reported worldwide, some cases remain undetected or neglected.<a class="elsevierStyleCrossRef" href="#bib0035"><span class="elsevierStyleSup">7</span></a> Key approaches for preventing brucellosis in humans include controlling the disease in animals using epidemiological studies to assess diversity among strains, and estimating the epidemiological relationship between isolates from different geographical origins.<a class="elsevierStyleCrossRefs" href="#bib0040"><span class="elsevierStyleSup">8,9</span></a></p><p id="par0010" class="elsevierStylePara elsevierViewall">Classical phenotyping methods, such as serotyping, phage typing, metabolic profiling and sensitivity to dyes have been employed for Brucella subtyping; however, these techniques are only available in reference laboratories, have a limited discriminatory power, require manipulation, and lack any standardized interpretation of this method cause difficulties.<a class="elsevierStyleCrossRef" href="#bib0050"><span class="elsevierStyleSup">10</span></a></p><p id="par0015" class="elsevierStylePara elsevierViewall">Based on these limitations, bacterial typing has shifted towards molecular identification by which epidemiological relationships are investigated among isolates. Thus far, several molecular typing methods, such as random amplified polymorphic DNA (RAPD)-PCR, amplified fragment-length polymorphism (AFLP), pulsed field gel electrophoresis (PFGE), polymerase chain reaction restriction fragment length polymorphism (RFLP), multiple-locus sequence typing (MLST) and multiple-locus VNTR (variable number tandem-repeats) analysis (MLVA) have been introduced. MLVA method is considered to be an effective and rapid tool for monitoring variations of copy numbers of tandem repeat units (TRs) with a high discriminatory power.<a class="elsevierStyleCrossRef" href="#bib0055"><span class="elsevierStyleSup">11</span></a> This method is not only used in outbreaks and epidemiological and trace-back investigations, but also in confirmatory laboratories or screening of food-borne acquired infections. TR sequences are multiple alleles that can be presented on a single locus and be easily determined through agarose electrophoresis or capillary electrophoresis based on their size differences.<a class="elsevierStyleCrossRef" href="#bib0060"><span class="elsevierStyleSup">12</span></a> MLVA has been proven to be a good technique for the assessment of pathogenic bacteria, such as Brucella, that display very little genomic diversity. MLVA schemes with 21, 15 and 16 loci (MLVA-21, MLVA-15 and MLVA-16, respectively) have been presented for discriminating Brucella spp. MLVA-16 has been proposed by Al-Dahank et al. with eight minisatellite markers (panel1: Bruce-06, Bruce-08, Bruce-11, Bruce-12, Bruce-42, Bruce-43, Bruce-45, and Bruce-55) for identification of bacteria spp and eight microsatellite markers (panel 2A: Bruce-18, Bruce-19, Bruce-21 and panel 2B: Bruce-04, Bruce-07, Bruce-09, Bruce-16 and Bruce-30) for screening the most common genotypes and classification of isolates to subspecies.<a class="elsevierStyleCrossRef" href="#bib0065"><span class="elsevierStyleSup">13</span></a> The genetic diversity of Brucella isolated from human and animal specimens has not yet been investigated in Iran. The main objectives of this study was to employ the MLVA-16 assay to investigate and assess the diversity of Brucella isolates for epidemiological purposes and to determine the most common genotypes of Brucella isolates in Iran.</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0045">Methods</span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0050">Ethical statement</span><p id="par0020" class="elsevierStylePara elsevierViewall">Ethical Committee of Iran University of medical science approved this study under the ethics code R.IUMS.REC 1396.33003.</p></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0055">Study design and sample size</span><p id="par0025" class="elsevierStylePara elsevierViewall">This study is a molecular-based and cross-sectional study to evaluate the possible reservoirs of human brucellosis in Iran.</p></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0060">Isolation and determination of Brucella spp</span><p id="par0030" class="elsevierStylePara elsevierViewall">The clinical samples were collected from humans and animals suffering from brucellosis. Culture isolation of Brucella spp was achieved by agar supplemented with 10% horse serum. The inoculated plates were incubated at 37 °C aerobically for 3–5 days. Single colonies were analyzed microscopically using Gram staining and biochemical (urease, oxidase and catalase) and motility tests were carried out for bacterial identification. Colonies confirmed as Brucella spp were subjected to PCR methods for species-level identification.</p></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0065">Preparation of Brucella DNA and amplification of loci</span><p id="par0035" class="elsevierStylePara elsevierViewall">DNA of Brucella was extracted using the PCR template preparation Kit (Roche Diagnostics, Germany). MLVA genotyping was performed as previously described by Flech et al. and completed by Al Dahouk et al. This method includes three panels named panel1, panel 2A and panel 2B. The 16 primer pairs were divided into three groups. Panel1 consists of eight minisatellite loci (Bruce 06, 08, 11, 12, 42, 43, 45, 55), while panel 2A (Bruce18, 19, 21) and panel 2B (Bruce 04, 07, 09, 16 and 30) consisted of three and five minisatellite loci, respectively. PCR amplification was performed in a total volume of 15 µl containing 1 ng of DNA, 1X PCR reaction buffer, 1U of Taq DNA polymerase, 200 mM of each deoxynucleotide triphosphate, and 0.3 mM of each flanking primer. Amplification was performed in an Eppendrof termocycler as follow: an initial denaturation step at 96 °C for 5 min, followed by 30 cycles of denaturation at 96 °C for 30 s, annealing at 60 °C for 30 s, and elongation at 70 °C for 1 min. The final extension step was performed at 70 °C for 5 min. Five microliters of amplification product was loaded on 2.5% agarose gel and run under a voltage of 8 V/cm. Gel images were then recorded.</p></span><span id="sec0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0070">Analysis of MLVA data</span><p id="par0040" class="elsevierStylePara elsevierViewall">The sizes of PCR products were analyzed and converted to repeat unit numbers using a published allele numbering system and the data set was imported to Bionumerics. For quantification of polymorphism at each locus, Hunted and Gaston diversity index (HGDI), available on the human protein HPA website (<a href="http://www.hpa-bioinformatics.org.uk/cgi-bin/DICI/DICI.pl">http://www.hpa-bioinformatics.org.uk/cgi-bin/DICI/DICI.pl</a>) was used. Categorical coefficient and unweighted pair group methods were applied for clustering analysis. Minimum spanning trees were also constructed.</p></span><span id="sec0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0075">Statistical analysis</span><p id="par0045" class="elsevierStylePara elsevierViewall">Statistical analyses were conducted using the SPSS version 18, (Inc, Chicago, IL, USA).</p></span></span><span id="sec0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0080">Results</span><span id="sec0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0085">Characteristics of patients and Brucella isolates</span><p id="par0050" class="elsevierStylePara elsevierViewall">A total of 54 Brucella spp. were respectively isolated from blood (62.96%) and cerebrospinal fluid (3.70%) of humans, as well as blood (16.66%), spleen (5.55%) and liver (11.11%) of animals. Human patients included 26 men (48.14%) and 10 women (18.51%) and animals included 15 sheep (27.77%) and three cattle (5.55%). Isolates were collected from three states of Iran, including Hamedan (59.25%), Arak (7.40%), and Tehran (33.33%). All isolates were identified either as <span class="elsevierStyleItalic">Brucella melitensis</span> or <span class="elsevierStyleItalic">Brucella abortus</span>. Three isolates (5.55%) were identified as <span class="elsevierStyleItalic">Brucella abortus</span> and 51 isolates (94.44%) were identified <span class="elsevierStyleItalic">Brucella melitensis</span>. The mean age of the 36 patients was 45, ranging from 6 to 80 years-old and male to female ratio was 2.6.</p></span><span id="sec0055" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0090">MLVA-16 genotyping results</span><p id="par0055" class="elsevierStylePara elsevierViewall">The complete MLVA16 assay was performed targeting panel 1, 2A and 2B loci. The repeat unit size of panel 1 loci was ≥ 9 bp, while that of panel 2A and 2B was up to 8 bp. The PCR products for 16 loci were converted to copy number of TRs. Based on the published data, the polymorphism of each 16 VNTR loci was analyzed by the MLVA-16 method. Based on this method 54 isolates with genetic similarity coefficient of 80% were divided into 46 genotypes. Of those, 22 genotypes were singletons, while 4, 2, 1, and 2 genotypes were represented by 2, 3, 4, and 7 isolates, respectively (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>). The most prevalent genotype was revealed in 14 strains. There were two more frequent genotypes, each observed in 7 isolates, one genotype was restricted to one geographic region (Hamadan), whereas the other was present in two regions (Hamadan and Tehran). Genotypes present in two or three isolates were from Tehran and Hamadan provinces (<a class="elsevierStyleCrossRef" href="#fig0010">Fig. 2</a>). Discriminatory power was determined for each studied locus in this study and according to the results, panel 2B showed the highest discriminatory power [ Bruce-04 (0.837), Bruce-30 (0.806), Bruce-09 (0.787), Bruce-07 (0.772) and Bruce16-(0.766)] while panel 2A displayed a moderate variability [Bruce-18 (0.805), Bruce-19 (0.568), and Bruce-21 (0.358)], and panel 1 demonstrated a limited diversity [Bruce-06 (0.204), Bruce-08 (0.654), Bruce-11 (0.673), Bruce-12 (0.598), Bruce-42 (0.408), Bruce-43 (0.541), Bruce-45 (0.713), Bruce-55 (0.730)].</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia><elsevierMultimedia ident="fig0010"></elsevierMultimedia></span></span><span id="sec0060" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0095">Discussion</span><p id="par0060" class="elsevierStylePara elsevierViewall">In the current study a total of 54 Brucella isolates were collected from human and animal samples from three regions of Iran and MLVA-16 was used to assess the genetic diversity among the isolates. Thirty-six (66.66%) isolates were from humans and 18 (33.33%) from animals. Three isolates (5.55%) were identified as <span class="elsevierStyleItalic">Brucella abortus</span> and the remaing 51 were <span class="elsevierStyleItalic">Brucella melitensis</span>. Brucellosis is an atypical zoonotic disease and constant monitoring of animals will largely contribute to the control human brucellosis.<a class="elsevierStyleCrossRefs" href="#bib0070"><span class="elsevierStyleSup">14,15</span></a> Human brucellosis is most often linked with animal husbandry or consuming unpasteurized milk.<a class="elsevierStyleCrossRefs" href="#bib0080"><span class="elsevierStyleSup">16–18</span></a> MLVA-16 is a suitable typing method for establishing an epidemiological relationship between isolates in an outbreak to avoid excessive classical epidemiological investigations.<a class="elsevierStyleCrossRefs" href="#bib0095"><span class="elsevierStyleSup">19,20</span></a> MLVA-16 with a genetic similarity coefficient of 0.8, yielded a total of 46 genotypes, among which 22 were singletons. The frequency of different MLVA genotypes varied in the three studied regions. Three B. melitensis isolates collected from two sheep samples (blood and spleen), one isolates from cattle (liver) and one from human (CSF) in Arak were distinctive in genotypes. Two B. abortus isolated from human blood and one from cattle blood was also distinctive in genotypes. The results of this study showed no correlation between the genotypes of B. abortus isolated from cattle and those isolated from men.</p><p id="par0065" class="elsevierStylePara elsevierViewall">Fifty B. melitensis isolates from human blood in Hamadan shared the same genotype as B. melitensis isolates from sheep blood and B. abortus isolates from cattle spleen in Hamadan, suggesting a possible epidemiological connection among them. These results were consistent to the study of Foster et al. who reported the spread of <span class="elsevierStyleItalic">Brucella</span> from animals to other animals or humans, and the epidemiologic relationship among them.<a class="elsevierStyleCrossRef" href="#bib0101"><span class="elsevierStyleSup">21</span></a> Four other genotypes isolated from human blood in Hamedan were very closely related and differed by a single repeat unit at one or two of the most variant loci. Variety in single or double loci may reflect the microevolution of B. melitensis isolates. Additionally, three B. melitensis strains isolated from the blood of three human patients in Hamadan and Tehran, two B. melitensis strains isolated from the blood of animals, two isolates from blood of a human patient and one B. melitensis strain isolated from the liver of sheep were completely matched. These results suggest transmission of Brucella from animals to humans. There are some control measurements and financial compensation for the slaughtered seropositive cattle. However, no such measurement exists for sheep or goat, and therefore, sheep infected with Brucella are one of the main sources for human and animal brucellosis in Iran. This finding is similar to that reported in study from Hai Jiang, China<a class="elsevierStyleCrossRefs" href="#bib0102"><span class="elsevierStyleSup">22,23</span></a>. Similar to previous studies, the highest diversity discriminatory power was found for panel 2B (0.7936) followed by panel 2A (0.557) and panel 1 (0.4760). According to these results, the loci of panel 1 including minisatellite loci with repeat unit length above 9 bp are more conserved than those in panel 2A and panel 2B with more heterogeneous microsatellite loci. The highest discriminatory power was indicated for Bruce30 (0.806) of panel 2B followed by Bruce18 (0.805) of panel 2A.</p></span><span id="sec0065" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0100">Conclusion</span><p id="par0070" class="elsevierStylePara elsevierViewall">The MLVA16 analysis on 54 Brucella isolates showed high polymorphism of genotypes. Only two genotypes, each observed in seven isolates, were related to one another and only one of these genotypes belonged to two separate regions.</p></span><span id="sec0070" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0105">Availability of data and materials</span><p id="par0075" class="elsevierStylePara elsevierViewall">Data are available upon request from corresponding author.</p></span><span id="sec0075" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0110">Authors' contributions</span><p id="par0080" class="elsevierStylePara elsevierViewall">SM initiated and supervised the study. SM and FM designed the experiments. FM and AN conducted the experiments. AN conducted the analysis. SM, FM and AN has written the manuscript. All authors have read and approved the manuscript.</p></span><span id="sec0080" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0115">Funding</span><p id="par0085" class="elsevierStylePara elsevierViewall">There is no funding for the present study.</p></span><span id="sec0085" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0120">Ethics approval and consent to participate</span><p id="par0090" class="elsevierStylePara elsevierViewall">This study was carried according to approved protocol by Ethics committees of Iran University of medical sciences.</p></span><span id="sec0090" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0125">Consent for publication</span><p id="par0095" class="elsevierStylePara elsevierViewall">Not applicable.</p></span><span id="sec0095" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0130">Competing interests</span><p id="par0100" class="elsevierStylePara elsevierViewall">The authors declare that they have no competing interests.</p></span></span>" "textoCompletoSecciones" => array:1 [ "secciones" => array:16 [ 0 => array:3 [ "identificador" => "xres1494418" "titulo" => "Abstract" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "abst0005" "titulo" => "Background" ] 1 => array:2 [ "identificador" => "abst0010" "titulo" => "Methods" ] 2 => array:2 [ "identificador" => "abst0015" "titulo" => "Results" ] 3 => array:2 [ "identificador" => "abst0020" "titulo" => "Conclusion" ] ] ] 1 => array:2 [ "identificador" => "xpalclavsec1356879" "titulo" => "Keywords" ] 2 => array:2 [ "identificador" => "xpalclavsec1356880" "titulo" => "Abbreviations" ] 3 => array:2 [ "identificador" => "sec0005" "titulo" => "Introduction" ] 4 => array:3 [ "identificador" => "sec0010" "titulo" => "Methods" "secciones" => array:6 [ 0 => array:2 [ "identificador" => "sec0015" "titulo" => "Ethical statement" ] 1 => array:2 [ "identificador" => "sec0020" "titulo" => "Study design and sample size" ] 2 => array:2 [ "identificador" => "sec0025" "titulo" => "Isolation and determination of Brucella spp" ] 3 => array:2 [ "identificador" => "sec0030" "titulo" => "Preparation of Brucella DNA and amplification of loci" ] 4 => array:2 [ "identificador" => "sec0035" "titulo" => "Analysis of MLVA data" ] 5 => array:2 [ "identificador" => "sec0040" "titulo" => "Statistical analysis" ] ] ] 5 => array:3 [ "identificador" => "sec0045" "titulo" => "Results" "secciones" => array:2 [ 0 => array:2 [ "identificador" => "sec0050" "titulo" => "Characteristics of patients and Brucella isolates" ] 1 => array:2 [ "identificador" => "sec0055" "titulo" => "MLVA-16 genotyping results" ] ] ] 6 => array:2 [ "identificador" => "sec0060" "titulo" => "Discussion" ] 7 => array:2 [ "identificador" => "sec0065" "titulo" => "Conclusion" ] 8 => array:2 [ "identificador" => "sec0070" "titulo" => "Availability of data and materials" ] 9 => array:2 [ "identificador" => "sec0075" "titulo" => "Authors' contributions" ] 10 => array:2 [ "identificador" => "sec0080" "titulo" => "Funding" ] 11 => array:2 [ "identificador" => "sec0085" "titulo" => "Ethics approval and consent to participate" ] 12 => array:2 [ "identificador" => "sec0090" "titulo" => "Consent for publication" ] 13 => array:2 [ "identificador" => "sec0095" "titulo" => "Competing interests" ] 14 => array:2 [ "identificador" => "xack523335" "titulo" => "Acknowledgements" ] 15 => array:1 [ "titulo" => "References" ] ] ] "pdfFichero" => "main.pdf" "tienePdf" => true "fechaRecibido" => "2020-09-13" "fechaAceptado" => "2020-11-30" "PalabrasClave" => array:1 [ "en" => array:2 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Keywords" "identificador" => "xpalclavsec1356879" "palabras" => array:3 [ 0 => "VNTR" 1 => "MLVA16" 2 => "Brucella" ] ] 1 => array:4 [ "clase" => "abr" "titulo" => "Abbreviations" "identificador" => "xpalclavsec1356880" "palabras" => array:7 [ 0 => "RAPD-PCR" 1 => "AFLP" 2 => "PFGE" 3 => "MLST" 4 => "VNTR" 5 => "MLVA" 6 => "TRS" ] ] ] ] "tieneResumen" => true "resumen" => array:1 [ "en" => array:3 [ "titulo" => "Abstract" "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0010">Background</span><p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">Epidemiological studies are important tools to assess the diversity of Brucella isolates and to estimate their epidemiological relationship among isolates from different geographical origins. In this study the MLVA16 (multiple-locus variable number tandem repeat analysis based on 16 loci) was employed to investigate the diversity of Brucella spp. Isolated from humans and animals for epidemiological purposes and to determine the most common Brucella genotypes in Iran.</p></span> <span id="abst0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0015">Methods</span><p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">We designed a molecular-based study to evaluate the potential reservoirs of human brucellosis. After isolation and identification of 54 Brucella spp human and animal specimens from three regions of Iran, bacterial genomic DNA was extracted MLVA with three panel was used for the genotyping of isolates. The size of PCR products were analyzed and converted to repeat unit numbers using a published allele numbering system and data set was imported into Bionumerics.</p></span> <span id="abst0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0020">Results</span><p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">Three isolates (5.55%) were identified as <span class="elsevierStyleItalic">Brucella abortus</span> and 51 (94.44%) as <span class="elsevierStyleItalic">Brucella melitensis</span>. Two isolates of <span class="elsevierStyleItalic">Brucella abortus</span> were from humans and one from an animal. Thirty-four <span class="elsevierStyleItalic">Brucella melitensis</span> isolates were from humans and 17 from animals. Using MLVA16-genotyping, 54 isolates with genetic similarity coefficient of 80% were divided into 46 genotypes and 22 genotypes were represented by a single isolate, while 4, 2, 1 and 2 genotypes were represented by 2, 3, 4 and 7 isolates, respectively. The most prevalent genotype was represented by 14 isolates. There were two other frequent genotypes each represented by seven isolates, among which only one was restricted to a geographic region. Discriminatory power for each locus was determined in this study and panel 2B shows the high discretionary power [Bruce04 (0.837), Bruce30 (0.806), Bruce 09 (0.787), Bruce 07 (0.772), Bruce16 (0.766)].</p></span> <span id="abst0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0025">Conclusion</span><p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">MLVA16 analysis of 54 Brucella isolates showed high level polymorphism in their genotypes. Only two genotypes, each observed in seven isolates, were related to one another and only one of these genotypes were found in to two separate regions.</p></span>" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "abst0005" "titulo" => "Background" ] 1 => array:2 [ "identificador" => "abst0010" "titulo" => "Methods" ] 2 => array:2 [ "identificador" => "abst0015" "titulo" => "Results" ] 3 => array:2 [ "identificador" => "abst0020" "titulo" => "Conclusion" ] ] ] ] "multimedia" => array:2 [ 0 => array:8 [ "identificador" => "fig0005" "etiqueta" => "Fig. 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 3030 "Ancho" => 2917 "Tamanyo" => 825086 ] ] "detalles" => array:1 [ 0 => array:3 [ "identificador" => "at0005" "detalle" => "Fig. " "rol" => "short" ] ] "descripcion" => array:1 [ "en" => "<p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">Dendrogram based on MLVA-16 assays for 54 isolates of <span class="elsevierStyleItalic">Brucella melitensis</span> and <span class="elsevierStyleItalic">Brucella abortus</span>. The columns represent the number of strains, geographical origin and bacterial sources.</p>" ] ] 1 => array:8 [ "identificador" => "fig0010" "etiqueta" => "Fig. 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 2522 "Ancho" => 3000 "Tamanyo" => 294711 ] ] "detalles" => array:1 [ 0 => array:3 [ "identificador" => "at0010" "detalle" => "Fig. " "rol" => "short" ] ] "descripcion" => array:1 [ "en" => "<p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">Minimum spanning tree for <span class="elsevierStyleItalic">Brucella</span> isolates using MLVA-16 data.</p>" ] ] ] "bibliografia" => array:2 [ "titulo" => "References" "seccion" => array:1 [ 0 => array:2 [ "identificador" => "bibs0005" "bibliografiaReferencia" => array:23 [ 0 => array:3 [ "identificador" => "bib0005" "etiqueta" => "1" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "Genotypic Expansion Within the Population Structure of Classical Brucella Species Revealed by MLVA16 Typing of 1404 Brucella Isolates From Different Animal and Geographic Origins, 1974-2006" "autores" => array:1 [ 0 => array:2 [ "etal" => true "autores" => array:6 [ 0 => "G. 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| Year/Month | Html | Total | |
|---|---|---|---|
| 2024 October | 20 | 21 | 41 |
| 2024 September | 58 | 37 | 95 |
| 2024 August | 68 | 59 | 127 |
| 2024 July | 70 | 35 | 105 |
| 2024 June | 38 | 29 | 67 |
| 2024 May | 30 | 23 | 53 |
| 2024 April | 39 | 54 | 93 |
| 2024 March | 29 | 39 | 68 |
| 2024 February | 27 | 51 | 78 |
| 2024 January | 27 | 32 | 59 |
| 2023 December | 32 | 40 | 72 |
| 2023 November | 29 | 38 | 67 |
| 2023 October | 36 | 40 | 76 |
| 2023 September | 42 | 41 | 83 |
| 2023 August | 26 | 23 | 49 |
| 2023 July | 25 | 19 | 44 |
| 2023 June | 18 | 19 | 37 |
| 2023 May | 43 | 17 | 60 |
| 2023 April | 36 | 19 | 55 |
| 2023 March | 57 | 21 | 78 |
| 2023 February | 37 | 23 | 60 |
| 2023 January | 21 | 18 | 39 |
| 2022 December | 47 | 27 | 74 |
| 2022 November | 42 | 36 | 78 |
| 2022 October | 57 | 27 | 84 |
| 2022 September | 29 | 50 | 79 |
| 2022 August | 33 | 42 | 75 |
| 2022 July | 33 | 33 | 66 |
| 2022 June | 39 | 43 | 82 |
| 2022 May | 35 | 28 | 63 |
| 2022 April | 38 | 41 | 79 |
| 2022 March | 37 | 55 | 92 |
| 2022 February | 35 | 53 | 88 |
| 2022 January | 37 | 36 | 73 |
| 2021 December | 38 | 57 | 95 |
| 2021 November | 21 | 34 | 55 |
| 2021 October | 31 | 31 | 62 |
| 2021 September | 31 | 41 | 72 |
| 2021 August | 25 | 28 | 53 |
| 2021 July | 31 | 27 | 58 |
| 2021 June | 36 | 39 | 75 |
| 2021 May | 52 | 63 | 115 |
| 2021 April | 130 | 106 | 236 |
| 2021 March | 75 | 32 | 107 |
| 2021 February | 35 | 23 | 58 |
| 2021 January | 47 | 41 | 88 |
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