MLVA genotyping of Chinese human Brucella melitensis biovar 1, 2 and 3 isolates

<p>Abstract</p> <p>Background</p> <p>Since 1950, <it>Brucella melitensis </it>has been the predominant strain associated with human brucellosis in China. In this study we investigated the genotypic characteristics of <it>B. melitensis </it>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.</p> <p>Results</p> <p>A total of 105 <it>B. melitensis </it>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 <it>B. melitensis </it>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' <it>B. melitensis </it>group. Chinese <it>B. melitensis </it>are classified in limited number of closely related genotypes showing variation mainly at the panel 2B loci.</p> <p>Conclusion</p> <p>The 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 <it>B. melitensis</it>. 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.</p

Changes of predominant species/biovars and sequence types of Brucellaisolates, Inner Mongolia, China

BACKGROUND: Human brucellosis incidence in China was divided into 3 stages, high incidence (1950-1960s), decline (1970-1980s) and re-emergence (1990-2000s). Human brucellosis has been reported in all the 32 provinces, of which Inner Mongolia has the highest prevalence, accounting for over 40% of the cases in China. To investigate the etiology alteration of human brucellosis in Inner Mongolia, the species, biovars and genotypes of 60 Brucella isolates from this province were analyzed. METHODS: Species and biovars of the Brucella strains isolated from outbreaks were determined based on classical identification procedures. Strains were genotyped by multi locus sequence typing (MLST). Sequences of 9 housekeeping genes were obtained and sequence types were defined. The distribution of species, biovars and sequence types (STs) among the three incidence stages were analyzed and compared. RESULTS: The three stages of high incidence, decline and re-emergence were predominated by B. melitensis biovar 2 and 3, B. abortus biovar 3, and B. melitensis biovar 1, respectively, implying changes in the predominant biovars. Genotyping by MLST revealed a total of 14 STs. Nine STs (from ST28 to ST36), accounting for 64.3% of all the STs, were newly defined and different from those observed in other countries. Different STs were distributed among the three stages. ST8 was the most common ST in 1950-1960s and 1990-2000s, while ST2 was the most common in 1970-1980s. CONCLUSIONS: The prevalence of biovars and sequence types of Brucella strains from Inner Mongolia has changed over time in the three stages. Compared with those from other countries, new sequence types of Brucella strains exist in China

Abortion and various associated risk factors in dairy cow and sheep in Ili, China.

We studied livestock abortion and various associated risk factors in the Ili region of northwest China. Livestock abortion prevalence was estimated and correlated with infections (Brucellosis, Salmonellosis, Mycoplasma and Chlamydia seropositivity) and management (farming type and contact with other herds/flocks) risk factors. A total of 2996 serum samples (1406 cow, 1590 sheep) were identified by RBPT (Rose Bengal Plate Test) and c-ELISA (competitive-enzyme linked immunosorbent assay), and they showed the overall seroprevalence of brucellosis in the study area was cow 6.76%, sheep 9.50%. The seroprevalence of brucellosis in X county was cow 7.06%, sheep 9.12%; in H county was cow 11.70%, sheep 10.80%; and in Q county was cow 4.22%, sheep 9.11%. The overall seroprevalence of Mycoplasma in the study area was cow 3.20%, sheep 6.42%. The seroprevalence of Mycoplasma in X county was cow 3.39%, sheep 7.98%; in H county was cow 5.26%, sheep 9.97%; and in Q county was cow 2.11%, sheep 4.33%. The Odds ratio of brucellosis for cow and sheep, respectively, were 45.909 [95% CI 26.912-78.317, P<0.001] and 70.507 [95% CI 43.783-113.544, P<0.001] times higher than other abortion-related factors including mixed farming, contact with other flocks and Mycoplasma infection. A total of 54 samples, including aborted cow (22), sheep (30) fetuses and milk samples (2), were identified as Brucella melitensis (B. melitensis) positive. A total of 38 Brucella were isolated from 16 aborted cow, 20 sheep fetuses and 2 milk samples. All of these isolates were identified, and confirmed, as B. melitensis. A phylogenetic tree showed that the Brucella isolates closely matched the B. melitensis biovar 3 isolated in Inner Mongolia, China, and B. melitensis isolated from Norway and India. These results suggest that B. melitensis biovar 3 is the main pathogen responsible for cow and sheep abortion and also pose a human health risk. Additionally, livestock reproduction can also be influenced by Mycoplasma infection and managerial factors (farming type and contact with other herds/flocks), especially in remote areas

Comparative Genomic Analysis of <i>Brucella melitensis</i> Vaccine Strain M5 Provides Insights into Virulence Attenuation

<div><p>The <i>Brucella melitensis</i> vaccine strain M5 is widely used to prevent and control brucellosis in animals. In this study, we determined the whole-genome sequence of M5, and conducted a comprehensive comparative analysis against the whole-genome sequence of the virulent strain 16 M and other reference strains. This analysis revealed 11 regions of deletion (RDs) and 2 regions of insertion (RIs) within the M5 genome. Among these regions, the sequences encompassed in 5 RDs and 1 RI showed consistent variation, with a large deletion between the M5 and the 16 M genomes. RD4 and RD5 showed the large diversity among all <i>Brucella</i> genomes, both in RD length and RD copy number. Thus, RD4 and RD5 are potential sites for typing different <i>Brucella</i> strains. Other RD and RI regions exhibited multiple single nucleotide polymorphisms (SNPs). In addition, a genome fragment with a 56 kb rearrangement was determined to be consistent with previous studies. Comparative genomic analysis indicated that genomic island inversion in <i>Brucella</i> was widely present. With the genetic pattern common among all strains analyzed, these 2 RDs, 1 RI, and one inversion region are potential sites for detection of genomic differences. Several SNPs of important virulence-related genes (<i>motB</i>, <i>dhbC</i>, <i>sfuB</i>, <i>dsbAB, aidA, aroC</i>, and <i>lysR)</i> were also detected, and may be used to determine the mechanism of virulence attenuation. Collectively, this study reveals that comparative analysis between wild-type and vaccine strains can provide resources for the study of virulence and microevolution of <i>Brucella</i>.</p></div

Genotyping of human Brucella melitensis biovar 3 isolated from Shanxi Province in China by MLVA16 and HOOF.

BACKGROUND: Brucellosis presents a significant economic burden for China because it causes reproductive failure in host species and chronic health problems in humans. These problems can involve multiple organs. Brucellosis is highly endemic in Shanxi Province China. Molecular typing would be very useful to epidemiological surveillance. The purpose of this study was to assess the diversity of Brucella melitensis strains for epidemiological surveillance. Historical monitoring data suggest that Brucella melitensis biovar 3 is the predominant strain associated with the epidemic of brucellosis in Shanxi Province. METHODS/PRINCIPAL FINDINGS: Multiple-locus variable-number repeat analysis (MLVA-16) and hypervariable octameric oligonucleotide fingerprinting (HOOF-print) were used to type a human-hosted Brucella melitensis population (81 strains). Sixty-two MLVA genotypes (discriminatory index: 0.99) were detected, and they had a genetic similarity coefficient ranging from 84.9% to 100%. Eighty strains of the population belonged to the eastern Mediterranean group with panel 1 genotypes 42 (79 strains) and 43 (1 strain). A new panel 1 genotype was found in this study. It was named 114 MLVAorsay genotype and it showed similarity to the two isolates from Guangdong in a previous study. Brucella melitensis is distributed throughout Shanxi Province, and like samples from Inner Mongolia, the eastern Mediterranean genotype 42 was the main epidemic strain (97%). The HOOF-printing showed a higher diversity than MLVA-16 with a genetic similarity coefficient ranging from 56.8% to 100%. CONCLUSIONS: According to the MLVA-16 and HOOF-printing results, both methods could be used for the epidemiological surveillance of brucellosis. A new genotype was found in both Shanxi and Guangdong Provinces. In areas with brucellosis, the MLVA-16 scheme is very important for tracing cases back to their origins during outbreak investigations. It may facilitate the expansion and eradication of the disease

The differences between the whole genomes of M5, 16M, and other <i>Brucella</i> strains.

<p>Regions with higher and lower read coverage are identified by colored lines. Red: more than 4× means coverage, Orange: 2× to 4× means coverage, Blue: ¼ to ½× means coverage, and Aqua: less than ¼× means coverage. The window that is used to calculate the read coverage is 1000 bp with a 500 bp overlap (the figure will be indistinct with a smaller window); thus, only regions with lengths ≥ 1000 bp are displayed. This window is different from the method used to determine RDs.</p

Genes deleted from RDs of the <i>B. melitensis</i> vaccine strain M5.

*<p>CDM:Coverage Divided by Mean coverage.</p>*<p>RD1a: included in RD1.</p

Site-specific recombinase genes on <i>Brucella</i> strains.

<p>Site-specific recombinase genes on <i>Brucella</i> strains.</p

Genomic features of the newly sequenced genome of the <i>B. melitensis</i> vaccine strain M5 compared with the known genomic sequences of the virulent strain 16 M.

<p>Genomic features of the newly sequenced genome of the <i>B. melitensis</i> vaccine strain M5 compared with the known genomic sequences of the virulent strain 16 M.</p