Evaluation of a Multiplex PCR Assay (Bruce-ladder) for Molecular Typing of All Brucella Species, Including the Vaccine Strains

Abstract

An evaluation of a multiplex PCR assay (Bruce-ladder) was performed in seven laboratories using 625 Brucella strains from different animal and geographical origins. This robust test can differentiate in a single step all of the classical Brucella species, including those found in marine mammals and the S19, RB51, and Rev.1 vaccine strains.Brucellosis is caused by a facultative intracellular bacterium of the genus Brucella, and it is one of the most frequent bacterial zoonoses in low-income countries, where the control programs have not succeeded in eradicating this neglected zoonosis. The disease is a major cause of direct economic losses and an impediment to trade and exportation. The genus Brucella consists of six recognized species, designated on the basis of differences in pathogenicity and host preference: B. melitensis (goats and sheep), B. abortus (cattle and bison), B. suis (infecting primarily swine but also hares, rodents, and reindeer), B. ovis (sheep), B. canis (dogs), and B. neotomae (wood rats) (7). The discovery of Brucella in a wide variety of marine mammals has led to the proposal of two new species: B. ceti (cetaceans) and B. pinnipedialis (pinnipeds) (8). Some of these species include several biovars, which are currently distinguished from each other by an analysis of approximately 25 phenotypic characteristics, including requirement for CO 2 , H 2 S production, sensitivity to dyes and phages, and other metabolic properties (1). However, all these tests are time-consuming, require skilled technicians, and are not straightforward, and some reagents are not commercially available. In addition, handling of this microorganism represents a high risk for laboratory personnel, since most Brucella strains are highly pathogenic for humans. Accurate diagnostic and typing procedures are critical for the success of the eradication and control of the disease, and therefore the identification of the different species is of great epidemiological importance. In order to overcome most of these difficulties, PCR-based assays have been employed for molecular typing of Brucella species. However, one of the challenges of using DNA-based techniques for differentiating the various Brucella species and strains is their high degree of genetic homology (16). This article describes the evaluation of a new multiplex PCR assay (10), named Bruce-ladder, in seven different European laboratories. The PCR protocol was standardized previously (10), and the same protocol was used in all laboratories (see the supplemental material). The selection of the DNA sequences to design the PCR primers was based on species-specific or strain-specific genetic differences (Table 1). Each laboratory used its own Brucella strain collection, typed by standard bacteriological procedures (1). A total of 625 Brucella strains were used (see the complete list in the supplemental material). The collection included the reference strains of all biovars of B. abortus, B. melitensis, B. suis, and B. ovis, B. canis, B. neotomae, the B. abortus S19, B. ...

Brucellosis at the animal/ecosystem/human interface at the beginning of the 21st century

“…Real-time PCR assays based on some of the genetic markers described above were later developed for Brucella species identification (Al Dahouk et al, 2007c) but these tests have the same limitations regarding B. suis and B. abortus detection. AMOS-PCR provided the basis for other multiplex PCR assays, such as the Bruce-ladder-PCR, able to successfully discriminate isolates of all six classical species and the marine mammal brucellae (Lopez-Goni et al, 2008;Mayer-Scholl et al, 2010). These tests, which are remarkably robust and require no expensive laboratory equipment, display the expected specificity at species level, except for some strains belonging to the closely related B. canis and B. suis species .…”

Molecular Typing of Brucella melitensis and B. abortus from Human Blood Samples Using PCR-RFLP Method

“…Cloackaert et al ( 13 ) designed primers for amplification of omp2a and omp2b fragments and their sequences are shown in Table 1. Each PCR reaction mixture contained; 1X PCR buffer, 2 mM MgCl 2 , 1 μl template DNA (0.5 μg) , 0.15 mMdNTP, 2.5 U Taq DNA polymerase, 20 pmol of each forward and reverse primers and sterile distilled water up to 50 μl.…”

“…Several molecular typing methods are introduced to find DNA polymorphism that is able to identify the Brucella species and biovars, among which detection of polymorphisms by PCR -RFLP has several advantages including the easy implementation, interpretation and use for large quantities of samples (11)(12)(13). In this method, by omp25, omp2 and omp31 loci and all Brucella species can be differentiated and their biovars identified (14).…”

A rare case ofBrucella abortusendocarditis in South Africa

“…10,11 Valve dehiscence due to ring abscesses have been reported, as was the case in our patient. 2 The clinical features of Brucella endocarditis are indistinguishable from endocarditis caused by other more common organisms. Therefore, in a patient with a strong epidemiological link, a high degree of suspicion and subsequent appropriate diagnostic evaluation is essential.…”

“…Bruce-ladder Multiplex polymerase chain reaction confirmed that the isolate was Brucella abortus. 2 Oral doxycycline 100 mg twice daily and intravenous rifampicin 600 mg daily was commenced for a duration of 8 weeks. Gentamicin was continued for a total of 20 days.…”