Development of a new PCR assay to identify Brucella abortus biovars 5, 6 and 9 and the new subgroup 3b of biovar 3

One hundred twenty-nine Brucella field strains isolated from cattle in Cantabria, Spain, from March 1999 to February 2003, were analysed by using the AMOS-ERY PCR assay and by Southern blot hybridisation with a probe from insertion sequence IS711. Most of the field isolates produced only the ery band in the AMOS-ERY assay and showed a hybridisation pattern identical to that exhibited by reference strains of biovars 5, 6 and 9 of Brucella abortus, but different from strain Tulya, belonging to biovar 3 of B. abortus. However, typing of these strains by standard methods demonstrated that they belonged to biovar 3 of B. abortus. These results indicated that B. abortus biovar 3 was not genetically homogeneous and at least could be divided in two. In one class, that we called biovar 3a, would be the Tulya strain, while the local field strains would belong to biovar 3b. Cloning and nucleotide sequencing of a DNA fragment containing an IS711 copy exclusive of the B. abortus field strains from biovar 3b and reference strains from biovars 5, 6 and 9, revealed the existence of a 5.4 kb deletion close to an IS711 copy. Based on these data, we designed a new primer, which together with the IS711 AMOS primer produced a PCR fragment of 1.7 kb only from the isolates of biovars 3b, 5, 6 and 9 of B. abortus. No amplification products were produced with these primers from strains of the rest of species and biovars of Brucella and from bacteria phylogenetically close to Brucella analysed in this work. Addition of this primer to the AMOS-ERY PCR primer cocktail allows the positive distinction of B. abortus biovars 3b, 5, 6 and 9 from the rest of Brucella species and biovars.     

Characterisation of a new phage lytic for both smooth and non-smooth Brucella species

A brucella phage of the Izatnagar series, designated Iz1, was lytic for all Brucella species that are normally smooth, although the efficiency of plating varied between biovars and species. The phage was also lytic for rough strains of B melitensis and B suis and, to a lesser extent, B ovis. It displayed negligible lytic activity towards B canis and rough B abortus cultures. In its morphological and serological properties and its stability to inactivating agents, the Iz1 phage resembled other brucella phages.     

From the discovery of the Malta fever's agent to the discovery of a marine mammal reservoir, brucellosis has continuously been a re-emerging zoonosis

Brucellosis is not a sustainable disease in humans. The source of human infection always resides in domestic or wild animal reservoirs. The routes of infection are multiple: food-borne, occupational or recreational, linked to travel and even to bioterrorism. New Brucella strains or species may emerge and existing Brucella species adapt to changing social, cultural, travel and agricultural environment. Brucella melitensis is the most important zoonotic agent, followed by Brucella abortus and Brucella suis. This correlates with the fact that worldwide, the control of bovine brucellosis (due to B. abortus) has been achieved to a greater extent than the control of sheep and goat brucellosis (due to B. melitensis), these latter species being the most important domestic animals in many developing countries. The long duration and high cost of treatment of human brucellosis reduces the efficacy of the therapy. There is no human vaccine for brucellosis and the occurrence of brucellosis is directly linked to the status of animal brucellosis in a region. In this context, the Word Health Organization has defined the development of a human vaccine, besides the implementation of control and eradication programs in animals, as a high priority. The pathogenicity for humans of B. suis biovars 1, 3 and 4 is well established, whereas B. suis biovar 2 seems to be less pathogenic. Indeed, although hunters and pig farmers have repeatably experienced infectious contact with B. suis biovar 2 (found in wild boar and outdoor-rearing pigs in Europe), isolation of B. suis biovar 2 from human samples have only been seldom reported. Marine mammal brucellosis, due to two new proposed Brucella species i.e. B. cetaceae and B. pinnipediae, represents a new zoonotic threat but the pathogenicity for humans of the different Brucella species found in cetaceans and pinnipeds still has to be clearly established.     

Differential reactivity of bovine lymphocytes to species of Brucella

The reactivity of bovine lymphocytes to 4 species of Brucella was tested in thymidine-uptake assays, using long-term cultured lymphocytes and freshly obtained blood mononuclear cells. Lymphocytes were taken from cows that had been challenge exposed with a virulent strain of B abortus at midgestation. The cows were classified retrospectively as being naturally resistant or susceptible to brucellosis. Lymphocytes taken from these cows had 3 patterns of reactivity with species of Brucella: pattern 1 was defined by reactivity with 4 species (B abortus, B canis, B suis, and B melitensis); pattern 2 was defined by reactivity with all these species, except B melitensis; pattern 3 was defined by reactivity with B abortus and B canis, but not with B suis or B melitensis. There was a statistically significant correlation between susceptibility to brucellosis and expression of lymphocyte cross-reactivity with B suis (P less than 0.01) and with B melitensis (P less than 0.001).     

布鲁菌进化和分类学研究进展

布鲁菌属革兰氏阴性兼性胞内寄生菌,能感染多种宿主动物和人。该属可分为6个典型种,包括羊种、牛种、猪种、沙林鼠种、绵羊附睾种以及犬种布鲁菌等。此分类是基于其致病性以及宿主偏好性的差异划分。尽管6个种通过传统表型试验能区分,但布鲁菌种内采用DNA-DNA杂交证明DNA同源性高度一致（相似性大于90%）。因此有人提议布鲁菌由单一种组成,即布鲁菌属中只有羊种布鲁菌,其他种都是羊种菌的生物亚型之一。然而基于其他分子技术的基因分型表明其DNA多态性表现明显,说明目前对这个种的分型还是比较准确。而最近分离的海洋种布鲁氏菌分离株（鳍型和鲸型）采用传统分型标准和一些特异的分子标记也证明这种分型比较正确。本文对目前布鲁菌种属进化和分类学进行综述,希望对研究其进化和分类有所帮助。     

牛、羊布鲁菌多重PCR方法的建立与应用

根据布鲁菌属特异性基因BCSP31和布鲁菌种间特异性标志IS711插入序列,设计合成了3对引物,以牛种布鲁菌544A、104M和羊种布鲁菌16M基因组DNA为模板,通过优化反应条件,建立了可同时检测布鲁菌属、牛种布鲁菌和羊种布鲁菌的多重PCR方法。牛种布鲁菌可扩增出301和114 bp 2条带,羊种布鲁菌可扩增出301和253 bp 2条带,该方法对牛种布鲁菌544A和羊种布鲁菌16M混合DNA模板的最小检出量为100 pg,对大肠杆菌O157∶H7、小肠结肠炎耶尔森菌等15种参照菌的核酸扩增结果均为阴性。应用该方法对吉林省某牛场的106份粪便进行检测,虎红平板凝集试验作对照,结果PCR检测9份为阳性,且全为牛种布鲁菌阳性,对应的虎红平板凝集试验也为阳性。结果表明,建立的多重PCR方法具有良好的敏感性和特异性,为布鲁菌病的鉴别诊断提供了一种分子检测工具。     

Use of polymerase chain reaction to identify Brucella abortus strain RB51 among Brucella field isolates from cattle in Italy

Brucella abortus strain RB51, a rough mutant of the B. abortus 2308 virulent strain, was recently approved in the United States as the official vaccine for brucellosis in cattle. Following recent evidence of unauthorized use of RB51 vaccine in Italy, where the use of vaccines for brucellosis is no longer allowed, the suitability of an RB51-specific polymerase chain reaction assay for identifying the RB51 strain among Brucella field isolates from cattle in Italy was investigated. The oligonucleotide primers used in this study, belonging to a six-primer cocktail for Brucella species previously described by other authors, allowed the amplification of a 364-base pair (bp) fragment specific for RB51 and its parent strain 2308, and a 498-bp product specific for B. abortus. In addition, unresolved bands ranging from 600 to 700 bp were observed from RB51 strain. Brucella abortus biovars 1, 2 and 4 have only one specific sensitive 498-bp band. The B. abortus biovars 3, 5 and 6 did not give any signal. The 498-bp product from a reference Brucella strain was sequenced and submitted to EMBL with the accession number AJ271969 while the 364-bp fragment from RB51 strain was submitted to EMBL database with accession number AJ271968. The sequence studies confirmed the specificity of the detected fragments. No amplification was obtained by testing DNA from strains antigenically related to Brucella, such as Yersinia enterocolitica O:9, Escherichia coli O:157, Salmonella urbana and Pasteurella multocida. The results of this study indicate that this technique, in combination with specific serological tests, could be a useful diagnostic method to verify the use of RB51 vaccine and can contribute to the creation of a databank of circulating strains.     

中国牛种布鲁菌弱毒疫苗株A19 SNP位点的研究

为鉴别我国牛种布鲁菌弱毒疫苗株A19与野生菌株,运用生物信息学方法结合基因测序,对疫苗株A19基因组SNP位点分析筛选,选取其中部分SNP位点,通过与布鲁菌常见种、生物型标准参考菌株和弱毒疫苗株基因组SNP位置核苷酸测序比较,验证SNP位点的A19特异性。结果表明,共筛选获得A19基因组29个SNP位点,验证ClpX G825-C825、LysR A605-C605、Omp2b G503-A503这3个SNP位点为A19（或S19）特异,揭示了A19基因组SNP位点分布情况,为疫苗株 A19与野生菌株鉴别提供了分子依据。     

布鲁菌S2疫苗株免疫牛与牛种、羊种布鲁菌自然感染牛ELISA鉴别诊断方法的建立

为建立区分猪种布鲁菌S2疫苗株接种奶牛与布鲁菌自然感染奶牛,BLAST比对分析羊种、牛种、猪种、犬种、沙林鼠种和绵羊种6种布鲁菌基因序列,发现repA—related基因是猪种布鲁菌与牛种及羊种布鲁菌的差异基因。设计引物PCR扩增获得repA-related基因片段,克隆并原核表达得到了布鲁菌repA—related融合蛋白,以repArelated蛋白建立间接EI．IsA检测方法。用repA—related蛋白间接ELISA检测猪种s2疫苗株接种动物血清为阳性,检测牛种和羊种布鲁菌自然感染动物血清为阴性。repA—related蛋白间接EusA能从试管凝聚实验（SAT）及常规ELIsA检测阳性的奶牛血清样本中,区分出s2疫苗接种牛与牛种布鲁菌感染牛。     

多重PCR方法鉴别牛、羊、猪种布鲁氏菌株

用eri作为布鲁氏菌属特异性基因,以IS711基因拷贝数差异作为布鲁氏菌种间特异性标志,建立了鉴别牛、羊、猪种布鲁氏菌株的多重PCR方法。结果:牛种布鲁氏菌2308株扩增出大小为494 bp和178 bp的两条带,羊种布鲁氏菌M28株扩增出大小为733 bp和178 bp的两条带,猪种布鲁氏菌S1330株扩增出大小为285 bp和178 bp的两条带,均与预期吻合;而胸膜肺炎放线杆菌、多杀性巴氏杆菌、流产沙门菌、都柏林沙门菌、大肠杆菌均未扩增出任何条带。硫化氢和血清学试验结果也符合相应种布鲁氏菌的特点。结果表明,本研究的多重PCR方法可用于牛、羊、猪种布鲁氏菌株的快速鉴定。     

Development of a Brucella suis specific hybridisation probe and PCR which distinguishes B. suis from Brucella abortus.

A genomic library was prepared from Brucella suis DNA (MboI digested) and cloned into the BamHI site of pUC18. Colony hybridisation using a probe prepared from purified B. suis DNA labelled with alpha 32P was carried out to identify colonies of interest. About 20 colonies, which gave an intense signal upon hybridisation with whole B. suis genomic DNA as a probe, were selected. Because of the high degree of DNA homology between B. suis and Brucella abortus, a short probe was chosen as it would more likely give species specificity. Of seven fragments selected to probe whole B. suis, B. abortus, and Yersinia enterocolitica DNA, one was found to hybridise with B. suis only. The probe was sequenced in two directions and sense and anti sense primers of 25bp in length were chosen to yield a product of 421bp. After optimisation of the PCR, a product of 420bp was obtained with B. suis template DNA and two bands of 420 and 650bp were detected with B. abortus template DNA. This is the first reported PCR of the Brucella genome where a single pair of primers will discriminate between B. suis and B. abortus. No band was observed when the two primers were used to amplify E. coli, Y. enterocolitica, Enterobacter cloacae, Staphylococcus aureus, Streptococcus uberis, Corynebacterium bovis, or Serratia marcescens template DNA.     

The identity, distribution and epizootiological significance of brucella isolates in Australia, 1981 to 1985

Results listing the identification of brucella isolates received by the National Brucellosis Reference Centre, National Biological Standards Laboratory, Canberra from 1981 to 1985 are presented. The distribution of brucella species and biotypes is shown on a host and state basis. Cultures isolated in Australia were identified as Brucella abortus biotypes 1, 2 and 4, and Strain 19; B. suis biotype 1, and B. ovis. B. melitensis biotype 3 was recovered from man infected in the Mediterranean area. B. abortus biotype 1 was the most frequent isolate. Atypical cultures isolated from cattle included B. suis biotype 1, and erythritol-utilising mutants of Strain 19. The epizootiological implications of these findings are discussed in relation to their impact on the national campaign to eradicate bovine brucellosis.     

Restriction endonuclease analysis of Brucella abortus

The restriction endonuclease profiles of bacterial DNA from Brucella abortus isolates were evaluated. It was not possible to distinguish between vaccine strain 19 and virulent (biotype 1 and biotype 2) strains of B abortus. Restriction endonuclease analysis is therefore not a suitable epidemiological tool in bovine brucellosis investigations. The genetic homogeneity of the Brucella genus was reinforced by these findings.     

Genetic diversity at alkB locus in Brucella abortus

DNA polymorphism of the alkB gene, a DNA repair gene, was assessed by PCR on Brucella abortus biovars 1 (strains 99, S19, 45/20, RB51 and 2308), 3 (Tulya strain), 5 (B3196 strain) and 6 (870 strain). A DNA repetitive element, named IS711, was detected in all studied biovars 1 and its complete nucleotide sequence was determined. We found that the element in alkB gene, bounded by 14 bp imperfect inverted repeats (IRs), is 840 bp long and appears to duplicate a consensus target site, CTAG. Analysing its nucleotide sequence of both forward and reverse strands, more than 10 open reading frames (ORFs) were found. Two potential transposase coding regions were chosen comparing all possible ORFs with the database. Comparing IS711 elements isolated from Brucella species, including both those characterized in our work and the published ones, differences in length and in nucleotide composition were observed among Brucella species, members of the same species and within the same strain. Our results confirm the heterogeneity of IS711 elements in Brucella genus and suggest the possibility to use this element to assess gene and genome diversity and to identify new molecular markers for Brucella species.     

Serological cross-reactions between different Brucella species and Yersinia enterocolitica. Immunodiffusion and immunoelectrophoresis

By immunodiffusion and immunoelectrophoresis tests in agarose serological cross-reactions were demonstrated between Yersinia enterocolitica type IX and Brucella strains from four species (Brucella abortus, Brucella melitensis, Brucella suis and Brucella neotomae). No qualitative differences between these strains in their tendencies to cross-react with Yersinia enterocolitica type IX were observed. Brucella canis and Brucella ovis, which have nonsmooth colonial morphology, gave no demonstrable cross-reaction with Yersinia enterocolitica type IX.The results of absorption tests and qualitative staining reaction of the obtained precipitation lines suggest that the antigenic determinants common to Brucella and Yersinia enterocolitica type IX seemed to be associated with the outer layer and in the lipopolysaccharide complex of the respective bacteria. By immunodiffusion and immunoelectrophoresis it was possible to identify in hyperimmune sera those antibodies that derive from Brucella and Yersinia enterocolitica type IX.Keyword: serological cross-reaction, Brucella species, Yersinia enterocolitica serotype IX, immunodiffusion, immunoelectrophoresis     

2308、M28、S1330三株不同种布鲁氏菌的毒力测定

为了测定牛、羊、猪三株不同种布鲁氏菌参考强毒株的毒力,选择了牛种2308、羊种M28和猪种S1330株,分别用雌性豚鼠（Hartley）和雌性小鼠（Balb/c）对其毒力进行测定。豚鼠测毒试验中,用含不同菌数的菌液腹股沟皮下注射5只豚鼠,测定2308、M28、S1330菌株的豚鼠最小感染量（MID）,结果显示以上3种毒株对豚鼠的最小感染量分别为9 CFU、10 CFU和30CFU。小鼠测毒实验中,将2308、M28和S1330菌液按1×105CFU/0.2 mL/只腹股沟皮下注射小鼠各5只,2周后分别剖杀小鼠,取脾脏测定含菌量,平均脾含菌量分别为1676971、314765、83811CFU/g脾脏。豚鼠和小鼠测毒均显示牛种2308株毒力最强,羊种M28株次之,猪种S1330毒力最弱。本研究首次用豚鼠和小鼠同时测定了布鲁氏菌2308、M28、S1330株的毒力,补充了布鲁氏菌参考强毒株的毒力数据。     

Entry and intracellular localization of Brucella spp. in Vero cells: fluorescence and electron microscopy

Vero cells were inoculated with the six species of Brucella (B. abortus, B. melitensis, B. suis, B. neotomae, B. canis, and B. ovis) and examined by fluorescence and electron microscopy. All Brucella spp. were internalized by Vero cells. In all cells except those inoculated with B. canis, the numbers of intracellular brucellae increased with time after inoculation. Intracellular brucellae were first seen within phagosomes and phagolysosomes. Subsequent localization within cisternae of the rough endoplasmic reticulum was seen with all species of Brucella, except B. canis, which was restricted to phagolysosomes. Although rough brucellae were more adherent and entered a greater number of Vero cells, intracellular replication occurred in a larger percentage of cells with smooth rather than with rough brucellae. These results suggest that phagocytosed Brucella spp. are transferred 1) to cisternae of the rough endoplasmic reticulum, where unrestricted bacterial replication takes place; or 2) to phagolysosomes in which Brucella spp. fail to replicate. The various strains of Brucella spp. differ in their ability to induce their own transfer to the rough endoplasmic reticulum.     

Monoclonal antibodies to Brucella surface antigens associated with the smooth lipopolysaccharide complex

Hybridomas producing antibodies to determinants associated with the lipopolysaccharide (LPS) of Brucella abortus and B melitensis were obtained by polyethylene glycol fusion of the SP2/0 myeloma cell line with B lymphocytes harvested from a Sprague-Dawley-derived rat previously immunized with whole B abortus strain 1119 organisms. Two clones, BRU38 and BRU28 , were selected for their ability to react with whole B abortus organisms and purified smooth-LPS ( f5p ). The BRU38 monoclonal antibodies were absorbed with live, rough strain 45/20 and smooth strains of B abortus and B melitensis organisms, whereas only smooth strains absorbed the antibody activity from BRU28 . Complete inhibition of the monoclonal's activity could be achieved with crude smooth-LPS, a purified f5p fraction, and a water-soluble acid degraded polysaccharide. Absorption of BRU38 and BRU28 with rough Brucella LPS, polysaccharide-B antigen, keto- deoxyoctanoic acid, or with several sugars and fatty acids known to be components of the Brucella LPS complex had no effect on the monoclonals. The data indicate that antigenic determinants are associated with the smooth LPS complex, probably with the O-side chain, and are expressed patchwise and in different quantities on several strains of B abortus and B melitensis. The B abortus rough strain 45/20 contains surface determinants which lead to the agglutination of smooth strain 1119 organisms. The potential use of monoclonals in competitive enzyme-linked immunosorbent assays for diagnostic purposes is discussed.     

Comparison of protein components of different species and strains of Brucella

The proteins constituents of Brucella abortus, Brucella melitensis and Brucella ovis were analyzed SDS-PAGE. From the comparison appears that the three species of Brucella studied shows a different electrophoretic pattern specially at the level of small peptides. On the contrary when two strains of B. abortus are analyzed no differences can be noticed.     

Effect of polymyxin B and environmental conditions on isolation of Brucella species and the vaccine strain RB51

Brucella are resistant to polymyxin B (PB), but their relative susceptibility to PB and its derivative, colistin (COL) has not been rigorously or systematically studied. Comparative susceptibility of Brucella reference strains, vaccine strain RB51, and Brucella isolates from marine mammals to these two cationic peptides were determined by Etest. Vast differences among Brucella species were found in susceptibility to both PB and COL. Brucella demonstrated similar pattern of relative susceptibility to PB as that of COL, but they were less susceptible to COL. Both B. melitensis and B. suis were the least susceptible to polymyxins and rough strains were more susceptible to both PB and COL than the smooth except for the BvrR mutant. Strains were generally less susceptible to PB when cultured in CO(2) rather than ambient air; some became more susceptible in acidified medium. Results show that environment cultural conditions must be considered when selecting for CO(2)-independent strains of Brucella especially the vaccine strain RB51 on selective media containing PB. Our observations extend basic knowledge of the differential resistance of Brucella to polymyxins.