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Mycobacterium avium subsp. paratuberculosis, the etiological agent of paratuberculosis, affects a wide range of domestic ruminants and has been suggested to be involved in Crohn's disease in humans. Most available methods for identifying and differentiating strains of this difficult species are technically demanding and have limited discriminatory power. Here, we report the identification of novel PCR-based typing markers consisting of variable-number tandem repeats (VNTRs) of genetic elements called mycobacterial interspersed repetitive units (MIRUs). Eight markers were applied to 183 M. avium subsp. paratuberculosis isolates from bovine, caprine, ovine, cervine, leporine, and human origins from 10 different countries and to 82 human isolates of the closely related species M. avium from France. Among the M. avium subsp. paratuberculosis isolates, 21 patterns were found by MIRU-VNTR typing, with a discriminatory index of 0.751. The predominant R01 IS900 restriction fragment length polymorphism type, comprising 131 isolates, was divided into 15 MIRU-VNTR types. Among the 82 M. avium isolates, the eight MIRU-VNTR loci distinguished 30 types, none of which was shared by M. avium subsp. paratuberculosis isolates, resulting in a discriminatory index of 0.889. Our results suggest that MIRU-VNTR typing is a fast typing method that, in combination with other methods, might prove to be optimal for PCR-based molecular epidemiological studies of M. avium/M. avium subsp. paratuberculosis pathogens. In addition, presumably identical M. avium subsp. paratuberculosis 316F vaccine strains originating from the Weybridge laboratory and from different commercial batches from Mérial actually differed by one or both typing methods. These results indicate a substantial degree of genetic drift among different vaccine preparations, which has important implications for prophylactic approaches.

Background: The classification of Brucella into species and biovars relies on phenotypic characteristics and sometimes raises difficulties in the interpretation of the results due to an absence of standardization of the typing reagents. In addition, the resolution of this biotyping is moderate and requires the manipulation of the living agent. More efficient DNA-based methods are needed, and this work explores the suitability of multiple locus variable number tandem repeats analysis (MLVA) for both typing and species identification.

A total of 51 strains (including type, reference, vaccine, and field strains) representing all species and biovars of Brucella formed a single deoxyribonucleic acid-deoxyribonucfeic acid hybridization group (Sl nuclease method). Accordingly, we propose that only one species, Bruceila melitensis, be recognized in the genus. We recommend that other specific epithets formerly associated with the generic name Brucella be used in a vernacular form for biovar designation (e.g., Brucella melitensis biovar Abortus 1). Carmichael and Bruner 1968 (8, 20). These species were proposed on the basis of differential tests and different preferential hosts (8).The identification of these bacteria at the species level is difficult and best suited to reference laboratories.In 1968, Hoyer and McCullough (13,14) studied six strains of Brucella, one of each species, by deoxyribonucleic acid (DNA)-DNA hybridization, using the DNA-agar method and a filter method. These authors (13,14) found that their strains are highly related. Unfortunately, the work of Hoyer and McCullough (13, 14) had a limited impact on the taxonomy of the genus Brucella, probably because DNA hybridization technology and its interpretation had not reached the state of development which it now has, too few strains were studied, and only two of the strains were type strains (B. melitensis 16M and B. canis RM6/66).The purpose of the present study was to apply modern DNA-DNA hybridization methods to 51 strains (including type strains) representing all present species and biovars of Brucella. Our results give no support to the division of the genus Brucella into more than one species. Consequently, we propose that only one species, B. melitensis, be recognized in this genus. MATERIALS AND METHODSBacterial strains. The 51 strains used in this study are listed in Table 1. All strains were kept freeze-dried in the Brucella Culture Collection, INRA-Nouzilly , Monnaie, France. Cultures were grown on Trypcase Soy Agar (Bio-Merieux, Marcy l'Etoile, France) supplemented with 0.1% (wthol) yeast extract (Difco Laboratories, Detroit, Mich.) (TSAYE medium). For fastidious strains (B. abortus biovar 2 and African biovar 3 and B. ovis), sterile equine serum (BioMerieux) was added to TSAYE medium to a final concentration of 5% (vol/vol) (TSAYES medium). The strains were checked for purity and species and biovar characterization by using standard procedures (1).DNA preparation. For each strain, six Roux flasks containing 250 ml of TSAYE or TSAYES medium were inoculated * Corresponding author with a 5-ml suspension in saline (pH 6.8) made with a 1-day culture on TSAYE or TSAYES medium slopes. After incubation for 72 h at 37°C under air (supplemented with 10% [vol/vol] C 0 2 if required), the growth from the six Roux flasks was harvested with 120 ml of 0.05 M tris(hydroxy me thy1)-aminomethane-0.05 M e t hy lenediaminetetraacetic acid-0.1 M NaCl buffer (pH 8). This suspension was checked for purity by streaking onto TSAYE or TSAYES medium. Then 2.6 ml of a 25% (wthol) aqueous solution...

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. ...