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。     

The first detection of brucella canis in cattle in the Republic of Korea

Twenty mammary lymph node samples were collected from cattle on a farm in the Republic of Korea. These cattle were serologically negative for Brucella by tube agglutination test (≤ 1:50) and serum agglutination test (≤ 1:50). Out of 20 lymph node samples, two samples were positive for Brucella growth on Brucella agar as well as blood agar. Tests for urease, hydrogen sulphide and reactions against monospecific sera A and M indicated that these two isolates (No. 15 and 16) belong to the genus Brucella. Genus specific, AMOS (abortus, melitensis, ovis, suis) and Bruce-ladder multiplex polymerase chain reaction (PCR) assays confirmed the Brucella isolates as either a B. abortus or a B. canis strain. This is the first report of the occurrence of a B. canis infection in cattle in Korea. More survey data are needed to determine whether B. canis is a significant aetiology in the cases of cattle brucellosis in Korea.     

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).     

New Bruce-ladder multiplex PCR assay for the biovar typing of Brucella suis and the discrimination of Brucella suis and Brucella canis

Rapid and specific identification of Brucella suis at the biovar level is necessary because some of the biovars that infect animals are pathogenic for humans. None of the molecular typing methods described so far are able to discriminate B. suis biovars in a single test and differentiation of B. suis from Brucella canis by molecular approaches can be difficult. This article describes a new multiplex PCR assay, Suis-ladder, for fast and accurate identification of B. suis at the biovar level and the differentiation of B. suis, B. canis and Brucella microti. An advancement of the original Bruce-ladder PCR protocol which allows the correct discrimination of all known Brucella species is also described.     

Genomic fingerprinting and development of a dendrogram for Brucella spp. isolated from seals, porpoises, and dolphins.

Genomic DNA from reference strains and biovars of the genus Brucella was analyzed using pulsed-field gel electrophoresis (PFGE). Fingerprints were compared to estimate genetic relatedness among the strains and to obtain information on evolutionary relationships. Electrophoresis of DNA digested with the restriction endonuclease XbaI produced fragment profiles for the reference type strains that distinguished these strains to the level of species. Included in this study were strains isolated from marine mammals. The PFGE profiles from these strains were compared with those obtained from the reference strains and biovars. Isolates from dolphins had similar profiles that were distinct from profiles of Brucella isolates from seals and porpoises. Distance matrix analyses were used to produce a dendrogram. Biovars of B. abortus were clustered together in the dendrogram; similar clusters were shown for biovars of B. melitensis and for biovars of B. suis. Brucella ovis, B. canis, and B. neotomae differed from each other and from B. abortus, B. melitensis, and B. suis. The relationship between B. abortus strain RB51 and other Brucella biovars was compared because this strain has replaced B. abortus strain 19 for use as a live vaccine in cattle and possibly in bison and elk. These results support the current taxonomy of Brucella species and the designation of an additional genomic group(s) of Brucella. The PFGE analysis in conjunction with distance matrix analysis was a useful tool for calculating genetic relatedness among the Brucella species.     

Brucella suis biotype 1 infection in a dog

Brucella suis biotype 1 was isolated from the semen of a dog with hindlimb weakness and a large, firm, left epididymis. A semen sample was oligospermic, with many neutrophils, the numbers of which decreased in serial sampling. A card agglutination test for B abortus and a rapid slide agglutination test for B canis were positive. The modified 2-mercaptoethanol slide agglutination test for B canis and the agar gel immunodiffusion test, using B canis cell wall antigen, were negative. At necropsy, chronic granulomatous inflammation was found in, and B suis biotype 1 was isolated from, the left epididymis and prostate gland.     

猪种布氏杆菌WboA基因缺失株对绵羊的免疫剂量及免疫程序研究

为优化猪种布氏杆菌WboA基因缺失株(B.suisΔWboA)对绵羊的免疫条件,本研究采用1岁左右的成年雌性绵羊对B.suisΔWboA的免疫剂量及免程序进行了比较研究。实验分5个组进行,其中A、B、C 3个试验组分别以2倍剂量重复接种、4倍剂量重复接种和单剂量1次接种B.suisΔWboA,D组单剂量1次接种猪种布氏杆菌S2疫苗株(B.suis S2),E组为空白对照组。各组羊首免后7 d、21 d和35 d分别采血,测定血清抗体水平;在首免后35 d,分别采用布氏杆菌强毒菌M28株(B.melitensis M28),经腹股沟皮下注射攻毒。攻毒后28 d,分别取试验羊的脾脏分离攻毒菌株。所有试验羊,在实施攻毒前,其精神、食欲均正常。血清抗体测定结果表明,在二免7 d、21 d后,A组和B组试验羊的抗体水平明显高于C组,而且均超过D组试验羊的抗体水平。攻毒后的细菌分离结果表明,攻毒后28 d,A组和B组试验羊的脾脏细菌分离数量明显低于C组试验羊,并且均低于D组试验羊的细菌分离水平。实验结果表明,B.suisΔWboA的免疫剂量由单倍改为2倍或4倍,免疫程序由单剂量1次改为2倍或4倍剂量2次,可以明显提高免疫效果,并达到与亲本疫苗菌株B.suis S2的免疫水平。     

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.     

Putative outer membrane autotransporter protein influences survival of Brucella suis in BALB/c mice

In Gram-negative bacteria, autotransporters are secreted proteins able to translocate themselves through the inner- and outer-membranes to the cell surface or to the extracellular environment. The influence of the putative outer membrane autotransporter (OmaA) protein to the persistence of Brucella suis was investigated. Sequence analyses revealed that the OmaA protein of B. suis strain 1330 consists of a signal peptide, a passenger alpha-domain, and a transporter beta-domain, which are the characteristic components of an autotransporter protein. The transporter beta-domain consists of 14 individual amphipathic beta-strands, and a 46-amino acid long alpha-helix lies upstream of the transporter domain, indicating that the B. suis OmaA is a type-I classical autotransporter. BLAST search and phylogenetic analyses revealed that the B. suis OmaA protein shares more similarities with adhesin autotransporter proteins than with protease autotransporter proteins of other bacteria. An OmaA-deficient strain (1330DeltaomaA) was generated by disrupting the DNA region encoding the passenger alpha-domain of the OmaA protein of B. suis wild type strain 1330. The omaA gene encoding the full-length OmaA protein was cloned and used to complement the OmaA-deficient strain. The OmaA-deficient strain did not differ from the wild type strain in terms of persistence in J774 macrophage cell line 24 and 48 h after inoculation, or clearance from the spleens of BALB/c mice at 1 week after intraperitoneal inoculation. These observations suggest that the function of the OmaA protein is dispensable during the acute phase of B. suis infection. However, the OmaA-deficient strain was cleared from the spleens of BALB/c mice faster than the wild type strain between the third and the ninth week after intraperitoneal inoculation, indicating that the OmaA may be important during the chronic phase of B. suis infection. Relative to the BALB/c mice injected with saline, those vaccinated with the OmaA-deficient strain exhibited 3.0-3.9log10 colony forming units protection against a challenge with B. suis strain 1330. This study is the first report correlating an autotransporter protein deficiency with persistence of B. suis in vitro and in vivo.     

The bovine immune response to Brucella abortus IV. Studies with a double immunodiffusion test for antibody against A2.

A double immunodiffusion test for precipitins against Brucella antigen A2 was developed and applied to a variety of samples. The A2 precipitins were produced by a heifer infected with B. abortus strain 2308, cattle vaccinated with killed B. melitensis strain H38 or live B. abortus strain 19 and by a dog infected with B. canis. Precipitins were also detected in the second International Standard for anti-Brucella abortus serum, in several anti-B. canis sera and at low levels in one anti-B. ovis serum tested. Antisera produced in calves against Yersinia enterocolitica serotype 0:9 had no anti-A2 activity despite titers greater than or equal to 1/1024 and greater than or equal to 1/80 in standard Brucella agglutination and CF tests, respectively. The test for A2 precipitins lacked specificity as weak reactions were obtained with five of 295 sera from brucellosis-free herds. This test was relatively insensitive, detecting precipitins in only 16 of 24 sera from infected cattle and 27 of 54 sera positive by complement fixation and enzyme labelled antiglobulin tests performed with whole cell and smooth lipopolysaccharide antigens, respectively. The A2 precipitins were detected in nine sera from five cattle, in two infected herds, which were negative by agglutination and complement fixation tests.     

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.     

Production and characterization of two Streptococcus suis capsular type 2 mutants.

Two avirulent mutants of Streptococcus suis capsular type 2 (M2 and M42) were produced from a highly virulent strain. Mutant M2, obtained after serial subcultures of the parent strain in the presence of rabbit anti-capsular type 2 serum, no longer possessed the type-specific capsular antigen, as demonstrated by serotyping methods and immunoelectron microscopy. The Lancefield group D antigen could not be detected on the cell surface of this mutant using the immunogold labelling technique. SDS-PAGE of lysozyme treated cells demonstrated that a 44 kDa protein which was present in the parent strain, was absent in mutant M2. Immunoblotting using rabbit whole cell homologous anti-serum revealed that the protein was strongly immunogenic. Mutant M2 was totally avirulent in mice, and the homologous antiserum completely failed to protect mice against challenge with the parent strain. However, mutant M42, obtained after passages of the parent strain at 42 degrees C, remained capsulated but lacked the same 44 kDa protein as mutant M2. The quantity of sialic acid present in the capsule was similar to that of the parent strain. Despite the presence of antibodies against the capsule, antiserum prepared against M42 only partially protected mice against a challenge with the parent strain. The 44 kDa cell wall protein could act as a virulence factor as well as an important immunogen of S. suis capsular type 2.     

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.     

一株犬种布鲁氏菌的分离与鉴定

对从一只流产贵宾犬全血中分离到的一株布鲁氏菌进行了形态结构、培养特性、生化特性研究以及凝集试验鉴定和多种属聚合酶链式反应(AMOS-PCR)鉴定.结果表明,分离菌为革兰氏阴性菌,在胰际琼脂培养基生长并呈典型的粗糙型布鲁氏菌菌落,菌落能被结晶紫溶液染成紫色.分离菌在琉瑾和复红培养基上生长,不产生H2S,与粗糙型布鲁氏菌阳性血清凝集,与光滑型布鲁氏菌阳性血清无凝集.用AMOS-PCR方法对分离菌进行鉴定,该分离菌具有犬种布鲁氏菌特有条带.鉴定结果表明分离菌符合犬种布鲁氏菌特性,将其命名为B.canis GB1.     

The bovine immune response to Brucella abortus. III. Preparation of antisera against a Brucella component precipitated by sera of some infected cattle.

Two methods are described for the partial purification of a high molecular weight, heat-resistant component (CO1) of sonicates of smooth and rough Brucella abortus which is precipitated by sera of some infected cattle. Method 1, a combination of gel filtration chromatography and polyacrylamide gel electrophoresis, was used to prepare CO1 from sonicates of a smooth field strain of B. abortus. Method 2, a combination of gel filtration chromatography and heat treatment, was used to obtain CO1, from sonicates of rough B. abortus strain 45/20. Rabbit antisera produced against CO1 prepared by either method contained only CO1 precipitins but were negative in standard agglutination and complement fixation tests conducted with whole cell antigens. Evidence is presented that CO1 is identical to Brucella antigen A2, and it is proposed that in future the designation A2 be employed.     

Protection of mice against Brucella abortus infection by inoculation with monoclonal antibodies recognizing Brucella O-antigen

Monoclonal antibodies recognizing the O-polysaccharide portion of Brucella abortus strain 2308 provided BALB/c mice with passive protection against challenge exposure with the homologous strain. Numbers of colony-forming organisms in the spleen were reduced by IgM and IgG monoclonal antibodies. Active immunization of mice, using B abortus 2308S lipopolysaccharide, resulted in production of IgM antibody at 14 days. Clearance of organisms in the actively immunized mice after challenge exposure at 14 days was nearly identical to that in passively immunized mice. Mice either passively or actively immunized were effectively protected from 0 to 28 days. Bacterial colonization of the spleen was observed to increase in both groups of mice at 56 days and indicated that humoral responses were effective in eliminating the organism in the early stages of infection, but other immune mechanisms were necessary for protection of mice in the later stage of infection with virulent strains of B abortus.     

犬种布氏菌地方株的DNA中G Cmol%测定

首次利用热变性法对9株地方犬种布氏菌DNA的G C含量进行了测定,同时以2株国际标准株为对照。G C mol％值在56.6～58.4范围内,与文献报道的犬种布氏菌和我们所用的2株标准株所得的G C mol％值是一致的。各株菌经常规检定、噬菌体裂解试验、血清学检查所得的结果均符合于犬种布氏菌的特性。说明本试验测定的G C mol％值代表了犬种布氏菌的DNA分子结构比值。为我国地方菌种、特别是粗糙型布氏菌的检定提供了一个新的方法。此外,对传统的DNA提取法进行了改进,使之更便于本试验的要求。     

犬布氏杆菌外膜蛋白Omp31基因的原核表达

克隆了犬布氏杆菌外膜蛋白Omp31基因并构建原核表达系统,并对表达产物进行了初步的血清学鉴定。利用PCR技术扩增犬布氏杆菌RM6/66参考株Omp31基因,然后将其克隆到pGEMT-easy载体上进行测序。测序正确后,将该基因插入到pET-32a载体中构建原核表达载体,转化大肠杆菌BL21感受态细胞,诱导表达融合蛋白,Western blot分析融合蛋白的免疫反应性。结果构建了犬布氏杆菌Omp31基因的原核表达载体pET-Omp31,并且在大肠杆菌中成功表达融合蛋白,经Western Blot鉴定该蛋白能被犬布氏杆菌阳性血清所识别。犬布氏杆菌外膜蛋白Omp31的表达成功,为犬布氏杆菌病血清学诊断方法的建立提供了基础资料。     

Effects of killed Brucella abortus strain 45/20 vaccine on reindeer later challenge exposed with Brucella suis type 4

Six seronegative pregnant reindeer (Rangifer tarandus L) were vaccinated with killed Brucella abortus strain 45/20 with added adjuvant. These were challenge exposed with B suis type 4 after 90 days; at the same time, 4 seronegative, nonvaccinated, pregnant reindeer (controls) were given the challenge inoculum. Humoral antibodies were detected in the vaccinated reindeer by postvaccination day 14. A marked increase in antibody levels also occurred after they were challenge exposed, but did not reach the levels observed in control reindeer which seroconverted within 8 days after they were given the challenge inoculum. One control reindeer aborted at 45 days after challenge exposure (at 165 days of a normal 225-day gestation period), and the fawn of another lived only a few days after delivery. Brucella suis type 4 was isolated from tissues of 3 of 4 control reindeer and of 2 of their fawns. All vaccinated reindeer gave birth to live fawns which were culture negative, although 1 fawn lived only a few days. Brucella suis type 4 was isolated from tissues of 1 vaccinated reindeer. Under the conditions of this experiment, killed B abortus 45/20 vaccine provided increased resistance to brucellosis.     

A rapid agglutination assay for canine brucellosis using antigen coated beads

Brucella canis is the causative agent of canine brucellosis and facultative intracellular pathogen. The diagnosis of canine brucellosis is based on bacteriological examination and serological methods including agglutination and gel diffusion tests. In this study, crude antigens were extracted from B. canis using hot saline, coated on to latex beads and their usefulness in the serological diagnosis of canine brucellosis was examined. Mixing the antigen coated latex beads with the sera of dogs infected with B. canis produced clear agglutination, but this was not so for B. canis free dog sera. N-terminal amino acid sequence analysis of the crude hot saline extracts, showed that they contained copper-zinc superoxide dismutase, ribose ABC transporter and hypothetical protein of Brucella as antigens. A serological survey of canine serum samples conducted by means of an agglutination test using the antigen coated latex beads, showed that this method was more specific than the tube agglutination test using whole bacterial cell antigens. Although these results suggest that our method in which crude hot saline extracted antigens are coated on to latex beads would be useful in the serological diagnosis of canine brucellosis, we need further investigation using more serum samples to confirm the usefulness of our method.