Molecular biological characteristics and differential diagnostic investigation of Brucella strains

Abstract

Brucellosis is one of the major zoonotic diseases that cause abortion, infertility in various animals and humans throughout the world. The causative agent of Brucella consists of ten species

six species (B. abortus, B. melitensis, B. suis, B. canis, B. ovis and B. neotomae) considered classical Brucella and four species (B. ceti, B. pinnipedialis, B. microti and B. inopinata) considered atypical Brucella. Besides, the genus expansion is still being processed, with the recent addition of B. papionis from baboons. Among them, three species are subdivided into biovars (bv.), i.e., B. abortus bv. 1-6 and 9, B. melitensis bv. 1-3, and B. suis bv. 1-5. In Korea, only B. abortus bv. 1 and 2, and B. canis have been isolated from livestock, wildlife and dogs. Currently, brucellosis is managed as Category II disease in Livestock Epidemics Prevention and Control Act and Group III contagious disease in Infectious Disease Prevention Act in Korea. To eradicate brucellosis, South Korea as well as many other countries has employed a strict test-and-slaughter strategy. As a result, since bovine brucellosis reached its peak in 2006, its prevalence has been decreased less than 0.1% in recent years. Meanwhile, Mongolia is still brucellosis-outbreak country and human brucellosis has the second-highest incidence in the world. So, lots of Brucella strains were obtained from various livestock, wildlife and humans. In this regard, it is a valuable work to characterize and apply them to evaluate new diagnostic methods. For effective prevention and control of brucellosis, an accurate identification and epidemiological trace-back analysis are required. In particular, the classification of species and biovars in Brucella is mainly depends on morphological, biological and biochemical characteristics. However, some B. abortus strains showed the unique phenotypic features which didn't match those of any biovars in the current taxonomy. Besides, these untyped strains demonstrated that they did not belong to bv. 1, 2 or 4 by additional molecular detections, such as species-specific PCR, BaSS-PCR and omp2a-PCR. Also, comparative 16S rRNA sequencing analysis revealed that in comparison to B. abortus reference strains (bv. 1-6 and 9), the untyped strains harbored distinct genetic traits. Taking these phenotyping and molecular typing results altogether, the untyped strains were suspected to be B. abortus bv. 7, which was suspended from the taxonomy in 1986. Our findings might be helpful to prove the existence of B. abortus bv. 7 and support the re-introduction into the Brucella taxonomy. For epidemiological analysis of Mongolian Brucella strains, the MLVA assay using 16 loci was performed. Mongolian Brucella strains were identified as B. abortus bv. 3 (9) and untype (6), and B. melitensis bv. 1 (67), bv. 3 (10) and Rev. 1 (17) by the classical biotyping. In the MLVA profiles, nine B. abortus bv.3 isolates were divided into four different genotypes, though six untype isolates were found to be in relation with only one genotype. The untyped B. abortus isolates predicted to be bv. 7, showed an identical MLVA profile with the Mongolian strains which were suggested as a reference bv. 7 strain in the recent report. In the MLVA assay including Web database, all sixty B. abortus strains were divided into two major clusters, and Mongolian B. abortus isolates were related with Chinese strains. In the MLVA-16 assay of B. melitensis strains, they were divided into 29 genotypes including four genotypes of B. melitensis Rev. 1 strains. As in the case of B. abortus, Mongolian B. melitensis also showed a high genetic similarity with Chinese strains as compared to other countries. To date, various diagnostic tools have been devloped to diagnose brucellosis, such as molecular detection and serologic tests using immunogenic antigens. For diagnosing of B. abortus infection with accuracy and rapidity, a new real-time PCR technique with a hybprobe from a specific SNP was developed. It could diagnose rapidly and exactly by an amplification curve monitoring and melting curve peak analysis. A total of 288 Brucella strains (22 reference strains, 156 Korean and 110 Mongolian isolates) and 8 non-Brucella were used to evaluate the diagnostic efficiency of the new PCR. With the regard to specificity, only B. abortus strains (bv. 1-6 and 9) revealed specific amplification curves from the 14th cycle, and Tm was 69 ºC in this PCR. In terms of sensitivity using B. abortus DNA and clinical samples, this new PCR was equal to or higher than that of 16S rRNA PCR. Therefore, it could be a useful molecular tool to diagnose B. abortus infection directly in animals and human due to its high sensitivity. As part of the development of serological assays, selection of highly immunogenic antigens is required for differential diagnosis. The current brucellosis serologic diagnosis is mainly based on detecting anti-LPS antibody, but it occasionally shows cross-reaction due to similar O-polysaccharide structure. To minimize cross-reaction, the B. abortus RB51 strain which is LPS-lacking mutant of the virulent B. abortus 2308 strain was used to search the immuno-dominant proteins by 2-DE and MALDI-TOF using four types of antisera. As a result, the identified eleven candidate antigens could be useful as alternative or supplementeal antigens for brucellosis diagnosis.