Evaluation and selection of tandem repeat loci for a Brucella MLVA typing assay

Flèche, Philippe Le; Jacques, Isabelle; Grayon, Maggy; Dahouk, Sascha Al; Bouchon, Patrick; Denœud, France; Nöckler, Karsten; Neubauer, Heinrich; Guilloteau, Laurence; Vergnaud, Gilles

doi:10.1186/1471-2180-6-9

Cited by 319 publications

(288 citation statements)

References 28 publications

(26 reference statements)

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“…One representative B. canis strain named bcanCR12 (here B. canis) isolated from a vaginal swab of a Pomeranian bitch after abortion was chosen for biological studies. The results of bacteriological analysis (33), Bruce-ladder multiplex PCR assay (31), multiplex SNP detection (32), and multilocus variable-number tandem-repeat analysis based on 16 loci (MLVA16) (34) were consistent with the B. canis genotype.…”

Section: Methodsmentioning

confidence: 91%

Brucella canis Is an Intracellular Pathogen That Induces a Lower Proinflammatory Response than Smooth Zoonotic Counterparts

et al. 2015

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e Canine brucellosis caused by Brucella canis is a disease of dogs and a zoonotic risk. B. canis harbors most of the virulence determinants defined for the genus, but its pathogenic strategy remains unclear since it has not been demonstrated that this natural rough bacterium is an intracellular pathogen. Studies of B. canis outbreaks in kennel facilities indicated that infected dogs displaying clinical signs did not present hematological alterations. A virulent B. canis strain isolated from those outbreaks readily replicated in different organs of mice for a protracted period. However, the levels of tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-12 in serum were close to background levels. Furthermore, B. canis induced lower levels of gamma interferon, less inflammation of the spleen, and a reduced number of granulomas in the liver in mice than did B. abortus. When the interaction of B. canis with cells was studied ex vivo, two patterns were observed, a predominant scattered cell-associated pattern of nonviable bacteria and an infrequent intracellular replicative pattern of viable bacteria in a perinuclear location. The second pattern, responsible for the increase in intracellular multiplication, was dependent on the type IV secretion system VirB and was seen only if the inoculum used for cell infections was in early exponential phase. Intracellular replicative B. canis followed an intracellular trafficking route undistinguishable from that of B. abortus. Although B. canis induces a lower proinflammatory response and has a stealthier replication cycle, it still displays the pathogenic properties of the genus and the ability to persist in infected organs based on the ability to multiply intracellularly. Brucellosis is a disease of animals and humans caused by members of the genus Brucella. Zoonotic species such as Brucella melitensis, Brucella abortus, and Brucella suis are facultative extracellular-intracellular stealthy pathogens that are able to overcome innate immunity at early times of infection (1-3) and at specific stages of adaptive immunity (4, 5). In addition to influencing the immune response, these Brucella species are able to circumvent the killing action of professional and nonprofessional phagocytes, transit within phagocytic vacuoles, and replicate extensively within the endoplasmic reticulum of cells (6). These properties allow the bacterium to spread throughout the reticuloendothelial system and promote chronic infection (3).There are other Brucella species that are also relevant pathogens; however, their infective strategies remain unclear and are not in tune with the solid results accepted for the previously mentioned zoonotic brucellae. Among these are Brucella canis, the etiological agent of brucellosis in dogs and a zoonotic pathogen (7). This pathogen induces a subclinical infection that may remain undiagnosed for protracted periods (8-10). B. canis invades the conjunctiva or the oronasal system or penetrates through the venereal route. Then it is distributed to different organs...

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Section: Methodsmentioning

confidence: 91%

Brucella canis Is an Intracellular Pathogen That Induces a Lower Proinflammatory Response than Smooth Zoonotic Counterparts

et al. 2015

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“…Two other studies used molecular analysis to identify more recent plasmodial DNA in ancient human remains, i.e., from 100-400 years ago (3,4). A substantial number of nonspecifi c amplifi cations in these previous studies raised concerns as to the specifi city of current molecular markers for ancient malaria (3,4).…”

Section: Plasmodium Falciparum In Ancient Egyptmentioning

confidence: 99%

“…Although it is believed that malaria widely affected early pre-Hippocrates populations, until now only 1 study, which used molecular analysis, clearly identifi ed P. falciparum in a Roman infant dating back to the 5th century AD (2). Two other studies used molecular analysis to identify more recent plasmodial DNA in ancient human remains, i.e., from 100-400 years ago (3,4). A substantial number of nonspecifi c amplifi cations in these previous studies raised concerns as to the specifi city of current molecular markers for ancient malaria (3,4).…”

Section: Plasmodium Falciparum In Ancient Egyptmentioning

confidence: 99%

“…from soil, 2 g each of 2 selected PCR-positive samples with the highest amplifi cation rate (both from the affected area) were thoroughly homogenized in 5 mL of phosphate-buffered saline (PBS), pH 7.2, in 50-mL tubes. To confi rm that strains BMS 17 and BMS 20 were B. microti, these strains were subjected to multilocus sequence analysis and multilocus variable number of tandem re-LETTERS peat analysis (MLVA) as described (1,(3)(4)(5) In summary, we successfully isolated B. microti from soil samples collected at the same site 7 years after primary isolation of this novel species from common voles. B. microti could still be isolated from the same soil samples 6 months after storage at 4°C.…”

Section: Isolation Of Brucella Microti From Soilmentioning

confidence: 99%

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Isolation ofBrucella microtifrom Soil

Scholz

Hubálek

Nesvadbová

et al. 2008

Emerg. Infect. Dis.

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al. Development of a real-time reverse transcriptase PCR assay for type A infl uenza virus and the avian H5 and H7 hemagglutinin subtypes. J Clin Microbiol. 2002;40:3256-60. DOI: 10.1128/JCM.40.9.3256-3260.2002 Isolation of Brucella microti from SoilTo the Editor: Brucella microti is a recently described Brucella species (1) that was isolated in 2000 from systemically infected common voles (Microtus arvalis) in South Moravia, Czech Republic. The organism is characterized by rapid growth on standard media and high metabolic activity, which is atypical for Brucella (2). The biochemical profi le of B. microti is more similar to that of Ochrobactrum spp., of which most species are typical soil bacteria.On the basis of the close phylogenetic relationship of Brucella spp. and Ochrobactrum spp. and the high metabolic activity of B. microti, we hypothesized that this Brucella species might also have a reservoir in soil. To test this hypothesis, we investigated 15 soil samples collected on December 11, 2007, from sites in the area where B. microti was isolated from common voles in 2000 (2). Ten of the samples were collected from the surface and at a depth of up to 5 cm near different mouse burrows 5 m apart. The remaining 5 samples were collected from an unaffected area without clinical cases of vole infection. The pH of soil samples ranged from 5.9 to 6.3. No frosts were recorded before the time of collection.To specifi cally detect B. microti in soil samples, we have developed a PCR that targets a genomic island of 11 kb (H.C. Scholz et al., unpub. data) that is unique for B. microti. Briefl y, primers Bmispec_f (5′-AGATACTGGAACATAGCCCG-3′) and Bmispec_r (5′-ATACTCAGGC AGGATACCGC-3′) were used to amplify a 510-bp fragment of the genomic island. PCR conditions were denaturation at 94°C for 5 min, followed by 29 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s. Total DNA was prepared from 0.5 g of each soil sample by using the MO BIO Ultra Clean Soil DNA Kit (Dianova, Hamburg, Germany). DNA was eluted with 50 μL of double-deionized water of which 2 μL was used in PCRs. Template DNA of B. microti CCM 4915T was used as a positive control. Type strains of all recognized Brucella species, 1 strain of each biovar of all species, and type strains of 11 Ochrobactrum species were used as negative controls.In this PCR, 5 of 15 soil samples and the positive control were positive for the 510-bp fragment; other Brucella spp. and Ochrobactrum spp. were negative. Of the 5 positive samples, 3 were collected from surface soil collected near mouse burrows. However, the remaining 2 positive samples were collected from the unaffected and supposedly negative-control area.For direct cultivation of Brucella spp. from soil, 2 g each of 2 selected PCR-positive samples with the highest amplifi cation rate (both from the affected area) were thoroughly homogenized in 5 mL of phosphate-buffered saline (PBS), pH 7.2, in 50-mL tubes. Of a serial dilution in PBS (10 0 -10 -4 ), 100 μL was plated onto Brucella agar (Merck, Darmstadt, German...

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“…Final biovar identification was performed by growth in the presence of thionine and basic fuchsin using the slide agglutination test with Brucella A-and M-mono-specific antisera (Veterinary Laboratories Agency, Weybridge, United Kingdom) (2). The same DNA samples were analyzed by the MLVA-16 typing technique, as described elsewhere (14,15), with some modifications. The 16 primer pairs were divided into two groups: panel 1 (loci Bruce06, Bruce08, Bruce11, Bruce12, Bruce42, Bruce43, Bruce45, and Bruce55) was more conserved and was characterized by moderately variable minisatellites, and panel 2 (loci Bruce04, Bruce07, Bruce09, Bruce16, Bruce18, Bruce19, Bruce21, and Bruce30) constituted highly discriminatory microsatellites (14,15).…”

mentioning

confidence: 99%

Link between Geographical Origin and Occurrence of Brucella abortus Biovars in Cow and Water Buffalo Herds

Borriello

Peletto

Lucibelli

et al. 2013

Appl Environ Microbiol

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c Sixty-three Brucella isolates from water buffaloes and cattle slaughtered within the Italian national plan for brucellosis control were characterized by multiple-locus variable-number tandem repeat analysis (MLVA). Genotyping indicated a strong influence of geographic origin on the Brucella abortus biovar distribution in areas where brucellosis is endemic and highlighted the importance of rigorous management procedures aimed at avoiding inter-and intraherd spreading of pathogens. Brucellosis is the most widespread zoonosis worldwide and is of major public health and economic significance (1). The disease is caused by Brucella spp., which can infect several important livestock species, including cattle, water buffaloes, goats, sheep, and pigs (1, 2). The principal symptom of the infection in all animal species is abortion or premature expulsion of the fetus. The pathogen can be transmitted to humans through consumption of contaminated and untreated milk or dairy products or by direct contact with infected animals. In humans, the disease can induce undulant fever, malaise, and myalgia, sometimes associated with serious complications, such as encephalitis, meningitis, peripheral neuritis, spondylitis, suppurative arthritis, and vegetative endocarditis. The disease can also occur in a chronic form that affects various organs and tissues (3). The genus Brucella includes 9 species (2) characterized by more than 90% DNA/ DNA homology (4, 5). In the last few years, the characterization of the variable number of tandem repeats (VNTR) by multiple-locus VNTR analysis (MLVA) was effectively used for typing of Brucella spp. in humans and animals, including bovine and ovine species, wild boars, hares, water buffaloes, and marine mammals (2, 4, 6, 7). Such typing of Brucella can be useful for epidemiological studies and may advance control of human and animal brucellosis.The two species most commonly involved in human infections are B. abortus, which is epizootic in cattle, and B. melitensis, which is more virulent and more diffused in sheep and goats (3). Brucellosis has been successfully eradicated in most developed countries, even though it is still endemic in many developing and some developed countries in Latin America, southern Europe, Africa, Southeast Asia, and the Middle East (1). The available strategies to control brucellosis are based on very strict management procedures, slaughter of all seropositive animals, and, where allowed, vaccination (8).The aim of this study was to evaluate the distribution of Brucella biovars in both cow and water buffalo herds in a region of brucellosis endemicity, to create a model of epidemiological traceback analysis useful to determine the origin of the contamination and consequently allow better management of the surveillance program (9).Sixty-three Brucella isolates obtained from lymph nodes of 46 water buffaloes and 17 cattle slaughtered within the Italian national plan for the control of brucellosis were investigated. As indicated by the Italian control program, based on a test...

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Evaluation and selection of tandem repeat loci for a Brucella MLVA typing assay

Cited by 319 publications

References 28 publications

Brucella canis Is an Intracellular Pathogen That Induces a Lower Proinflammatory Response than Smooth Zoonotic Counterparts

Brucella canis Is an Intracellular Pathogen That Induces a Lower Proinflammatory Response than Smooth Zoonotic Counterparts

Isolation ofBrucella microtifrom Soil

Link between Geographical Origin and Occurrence of Brucella abortus Biovars in Cow and Water Buffalo Herds

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