BackgroundSince 1950, Brucella melitensis has been the predominant strain associated with human brucellosis in China. In this study we investigated the genotypic characteristics of B. melitensis isolates from China using a multiple-locus variable-number tandem-repeat analysis (MLVA) and evaluated the utility of MLVA with regards to epidemiological trace-back investigation.ResultsA total of 105 B. melitensis strains isolated from throughout China were divided into 69 MLVA types using MLVA-16. Nei's genetic diversity indices for the various loci ranged between 0.00 - 0.84. 12 out 16 loci were the low diversity with values < 0.2 and the most discriminatory markers were bruce16 and bruce30 with a diversity index of > 0.75 and containing 8 and 7 alleles, respectively. Many isolates were single-locus or double-locus variants of closely related B. melitensis isolates from different regions, including the north and south of China. Using panel 1, the majority of strains (84/105) were genotype 42 clustering to the 'East Mediterranean' B. melitensis group. Chinese B. melitensis are classified in limited number of closely related genotypes showing variation mainly at the panel 2B loci.ConclusionThe MLVA-16 assay can be useful to reveal the predominant genotypes and strain relatedness in endemic or non-endemic regions of brucellosis. However it is not suitable for biovar differentiation of B. melitensis. Genotype 42 is widely distributed throughout China during a long time. Bruce 16 and bruce 30 in panel 2B markers are most useful for typing Chinese isolates.

In China, brucellosis is an endemic disease and the main sources of brucellosis in animals and humans are infected sheep, cattle and swine. Brucella melitensis (biovars 1 and 3) is the predominant species, associated with sporadic cases and outbreak in humans. Isolates of B. abortus, primarily biovars 1 and 3, and B. suis biovars 1 and 3 are also associated with sporadic human brucellosis. In this study, the genetic profiles of B. melitensis and B. abortus isolates from humans and animals were analyzed and compared by multi-locus variable-number tandem-repeat analysis (MLVA). Among the B. melitensis isolates, the majority (74/82) belonged to MLVA8 genotype 42, clustering in the ‘East Mediterranean’ group. Two B. melitensis biovar 1 genotype 47 isolates, belonging to the ‘Americas’ group, were recovered; both were from the Himalayan blue sheep (Pseudois nayaur, a wild animal). The majority of B. abortus isolates (51/70) were biovar 3, genotype 36. Ten B. suis biovar 1 field isolates, including seven outbreak isolates recovered from a cattle farm in Inner Mongolia, were genetically indistinguishable from the vaccine strain S2, based on MLVA cluster analysis. MLVA analysis provided important information for epidemiological trace-back. To the best of our knowledge, this is the first report to associate Brucella cross-infection with the vaccine strain S2 based on molecular comparison of recovered isolates to the vaccine strain. MLVA typing could be an essential assay to improve brucellosis surveillance and control programs.

In the present study, a total of 1102304 serum samples were collected to detected human brucellosis between the years 2012 and 2016 in Inner Mongolia. Overall, an average of 3.79% anti-Brucella positive in Inner Mongolia was presented but the range of positive rates were among 0.90 to 7.07% in 12 regions. Seroprevalence of human brucellosis increased gradually from 2012 to 2016. However, the incidence rate of human brucellosis showed a declining trend. One hundred and seven Brucella strains were isolated and identified as B. melitensis species, and B. melitensis biovar 3 was the predominant biovar. MLVA-11 genotypes 116 was predominant and had crucial epidemiology to the human population. All 107 strains tested were sorted into 75 MLVA-16 genotypes, with 54 single genotypes representing unique isolates. This result revealed that these Brucellosis cases had epidemiologically unrelated and sporadic characteristics. The remaining 21 shared genotypes among two to four strains, confirming the occurrence of crossinfection and multiple outbreaks. Extensive genotype-events were observed between strains from this study and Kazakhstan, Mongolia, and Turkey, these countries were key members of the grassland silk road. Long-time trade in small ruminants (sheep) in these countries has possibly promoted the spread of Brucella spp. in these regions.