Application and evaluation of the MLVA typing assay for the Brucella abortus strains isolated in Korea

Abstract

Background: A Brucella eradication program has been executed in Korea. To effectively prevent and control brucellosis, a molecular method for genetic identification and epidemiological trace-back must be established. As part of that, the MLVA typing assay was evaluated and applied to B. abortus isolates for analyzing the characteristics of the regional distribution and relationships of foreign isolates.

Results: A total of 177 isolates originating from 105 cattle farms for the period 1996 to 2008 were selected as representatives for the nine provinces of South Korea. A dendrogram of strain relatedness was constructed in accordance with the number of tandem repeat units for 17 loci so that it was possible to trace back in the restricted areas. Even in a farm contaminated by one source, however, the Brucella isolates showed an increase or decrease in one TRs copy number at some loci with high DI values. Moreover, those 17 loci was confirmed in stability via in-vitro and in-vivo passage, and found to be sufficiently stable markers that can readily identify the inoculated strain even if minor changes were detected. In the parsimony analysis with foreign Brucella isolates, domestic isolates were clustered distinctively, and located near the Central and Southern American isolates.

Conclusion: The MLVA assay has enough discrimination power in the Brucella species level and can be utilized as a tool for the epidemiological trace-back of the B. abortus isolates. But it is important to consider that Brucella isolates may be capable of undergoing minor changes at some loci in the course of infection or in accordance with the changes of the host.

Figure 1

The 177 prevalent B. abortus isolates from five loci appeared to be the allelic type. The most frequent allelic types in Bruce 04, Bruce 07, Bruce 30, Bruce 43, and Hoof 3, had four, four, six, three, and three copy numbers, respectively.

Figure 2

Cluster analysis for B. abortus isolates based on the dataset of 17 loci. Here was included in 105 B. abortus isolates (included RB51 isolate) and 11 B. abortus standard strains. All the isolates were confirmed to B. abortus strains and were classified into nine clusters and 23 genotypes (A1-I1). In the columns, the following data for isolates were given: species, biovar, strain ID, breed (Hanwoo; Korean native cattle), isolation year, farm, province, and district.

Figure 3

Geographic distribution of 104 B. abortus isolates from Korea. B. abortus isolates were selected in 104 outbreak farms (one strain/farm) from 1996 to 2008.

Figure 4

Maximum parsimony analysis of foreign B. abortus strains and Korean isolates. The data for 48 foreign strains including the reference strain were downloaded from the related websites http://mlva.u-psud.fr[23,30]. There were analyzed by 16 loci, except for Hoof 3, not as information of the foreign strains. The 23 Korean isolates, which were representing 23 genotypes, were compact and were located near the Central and Southern American isolates.

Figure 5

Variation of the B. abortus 2308 strains re-isolated via in-vivo passage in mice. Three of the 12 isolates were found to have increased to two TRs copy numbers at Hoof 3. In the rest of 16 loci, no change was detected. M, 25/100 bp ladder; 1, B. abortus 2308 strain; 2-13, B. abortus 2308 mouse passage isolates.