Isolation of Brucella abortus and Brucella abortus, strain 19, from cattle.

Tissues from 104 cows in herd were examined for brucellae. Brucella abortus, strain 19, was isolated from 22 cows, a field strain of B abortus, biotype 1, was isolated from 9 cows, and both strains were isolated from 2 cows.     

Monoclonal antibodies directed against Brucella abortus cell surface antigens

A panel of monoclonal antibodies (MAb) has been raised against Brucella abortus cell surface antigens from mice immunized with either heat/phenol treated or UV killed bacterial suspensions of B. abortus. The hybridomas were screened by either a microagglutination procedure or by an indirect enzyme immunoassay (EIA) on sonicated bacterial preparations. From a large number of MAb generated by various procedures, two distinct types of MAb emerged. The most numerous type was capable of agglutinating B. abortus and reacting with a soluble preparation of lipopolysaccharide (LPS). A second type was not capable of agglutinating the bacterial suspensions or of binding to the soluble LPS preparation but reacted with an antigen present in bacterial sonicates. Two MAb of this type react differentially with sonicates prepared from virulent and avirulent strains of B. abortus. There appeared to be sufficient evidence from our analysis of the relative degree of cross reaction with antigens present on a range of B. abortus strains and Brucella and xenogenic bacterial species to conclude that each of the seven MAb was recognising a separate antigenic site on the B. abortus cell surface.     

Molecular and cellular interactions between Brucella abortus antigens and host immune responses

Host protection against Brucella abortus, is thought to be mediated primarily by a Th1 type immune response. Unfortunately, only few specific bacterial antigens involved in stimulating protective cellular immunity against Brucella are known. Therefore, identifying bacterial proteins that induce a T-lymphocyte mediated response is critical to determine Brucella immunity. Several library screening methods are discussed that have been used to identify Brucella proteins that stimulate T lymphocytes including cellular immunoblotting, Escherichia coli expressed Brucella proteins, green fluorescence reporter systems, and signature tagged mutagenesis. Future studies would likely examine how bacterial proteins expressed within host cells aid pathogen survival and/or induce host responses. Some of these newly identified bacterial gene products may serve as antigens to activate a protective host immune response. Also, identifying Brucella proteins expressed at particular times during infection will also yield insights into Brucella pathogenesis.     

Characterization of salt-extractable protein antigens from Brucella abortus by crossed immunoelectrophoresis and isoelectricfocusing

Salt-extractable protein antigens (CSP) from Brucella abortus strains 19 and 2308 (vaccine and virulent strains, respectively) were analysed by crossed immunoelectrophoresis (CIE) using rabbit antisera to protein antigens and by isoelectricfocusing (IEF) in polyacrylamide gels. The reference immunoelectrophoretic profiles developed for proteins from strain 19 and 2308 of B. abortus contained 20 and 25 immunoprecipitates, respectively. Serum from cows experimentally infected or hyperimmunized with live organisms produced up to 5 immunoprecipitates in CIE with the protein antigens. Absorption of rabbit sera with homologous B. abortus cells reduced, but did not eliminate all of the immunoprecipitates from rabbit sera, suggesting that the majority, but not all of the protein components, are exposed on the surface of the cell. In contrast, antibody to protein antigens in agglutinin-free absorbed serum from infected cattle could still be demonstrated by CIE, even though CIE with protein extracts from whole cells radioiodinated with the cell surface labeling reagent, diazoiodusulfanilic acid, indicated that these antigens may be at or near the surface of the cell. From CIE in heterologous systems we concluded that all proteins present in strain 19 preparations were partially or completely identical to those in strain 2308. The IEF studies paralleled the CIE studies and revealed that the protein profile from strain 2308 was more complex than the profile from strain 19. Major differences between the 2 strains were found in the pH region from 3.9 to 5.0, where strain 2308 exhibited 4 additional protein bands.     

A serological comparison of complement fixation reactions using Brucella abortus and B. melitensis antigens in B. abortus infected cattle

Brucella abortus and B. melitensis antigens were used in parallel on the National Standard Brucella abortus antiserum and on field sera coming from cattle where practically exclusively B. abortus biotypes 1 and 2 have been isolated over the last 11 years. With the National Standard serum the titres to B. melitensis were consistently lower than those to B. abortus antigen. Most were 1 dilution (twofold) lower. Although a similar trend was seen with the field sera, there were 7/346 sera which had twofold or higher titres to B. melitensis antigen. Although this may be due to the vagaries of the test it also warrants closer investigation of the animals concerned to see whether M-antigen predominant Brucella biotypes are possibly present. The use of the dual antigens could identify herds which are infected only with A-antigen predominant brucellae but would not be reliable for classifying individual animals.     

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.     

Immunogenicity of Brucella abortus salt-extractable proteins

The immunogenic properties of salt-extractable proteins and chromatographic fractions thereof from Brucella abortus were evaluated in lemmings (Dicrostonyx rubricatus). The efficacy of the Brucella proteins as immunogens was determined after challenge with virulent B. abortus strain 2308 and was based on protection against clinical signs and gross lesions of brucellosis, as well as on numbers of viable Brucella in the spleen. Vaccination of lemmings with as little as 0.1 microgram of salt-extractable proteins (CSP) suppressed splenic infection, resulting in reduced numbers of viable organisms per spleen of 5-6 logs compared to non-vaccinated controls. Protein fractions separated by column chromatography were generally effective in reducing splenic infection, and contained proteins with molecular weights of 30,000, 20,000 and 12,000. Vaccines containing chemically modified dodecanoyl-CSP offered no additional advantage over unmodified CSP vaccines.     

Prevalence of Brucella abortus and Brucella canis antibodies in dogs in Nigeria

Serum samples collected from dogs brought for routine physical examination, vaccination and other complaints at the Small Animal Clinic of Ahmadu Bello University, Zaria, Nigeria were tested for Brucella abortus and Brucella canis antibodies. Ninety-five (38-2 per cent) of 249 dogs studied were positive for B. abortus agglutinins by the Rose Bengal plate test (RBPT) but none was sero-positive by the standard agglutination test (SAT). The antibody prevalence for B. canis by the SAT was 28-6 per cent for 224 dogs tested. Exotic breeds of dogs had a prevalence of 34-9 per cent for B. canis agglutinins while 28-1 per cent of local dogs were sero-positive. Twenty-two per cent of dogs older than 2 years were sero-positive compared to a prevalence of 33-3 per cent found amongst dogs younger than 1 year. A similar B. canis infection rate was observed amongst male (29-6 per cent) and female (26-7 per cent) dogs.     

牛布鲁氏菌膜蛋白的免疫抗原筛选

试验旨在通过免疫蛋白质组学筛选布鲁氏菌保护性抗原。利用双向电泳技术对试验条件下培养的布鲁氏菌强毒株544A膜蛋白进行分离,结合Western blotting技术分别用豚鼠、牛抗布鲁氏菌血清寻找发生免疫反应的蛋白质。16个免疫蛋白点经胶内酶切、质谱(LC-MS/MS)进行鉴定。利用生物信息学工具发现这些蛋白不全是膜蛋白,还存在胞质蛋白,其功能涉及生物合成和物质代谢等领域。成功建立了布鲁氏菌膜蛋白的免疫蛋白质组学研究方法,为寻找保护性抗原及为新型疫苗抗原候选提供新思路。     

Experimental infection of opossums with Brucella abortus

Thirteen opossums (Didelphis virginiana) trapped in east central Alabama were fed approximately 1.5 X 10(9) Brucella abortus colony forming units. Serologic responses to at least 1 of 3 tests developed in 8 of the 13 opossums. Brucella abortus was recovered from 18 of 159 blood samples from 4 of the 13 opossums and from 7 of 159 fecal samples from 6 of them. All culture-positive feces had been excreted within 4 days after exposure. Sixty-four urine, 123 saliva, and 78 vaginal samples were culture-negative. Eleven baby opossums, in their mothers' pouches at the time of capture, were culture-negative. Brucella abortus was isolated from 10 of 13 adult opossums. Ten additional opossums were trapped and tested for brucellosis. One had a tube agglutination titer of 1:25, and B abortus was isolated from the liver and spleen. Brucella abortus was isolated from lung and spleen of 8 seronegative opossums. The remaining 8 opossums were negative to all tests.     

Experimental infection of dogs with Brucella abortus

Thirteen female dogs, which included eight principals that were fed approximately 4.4 X 10(10) colony forming units (cfu) of Brucella abortus strain 2308 and five sentinels that were housed with the principals, were examined for serologic responses, blood culture, tissue distribution of the organisms and pathologic lesions. Serum samples from each dog were tested on the day of exposure and on post exposure days 5, 7, 10, 14, 21, 28, 35, 42 and 49 for antibodies to B. abortus, using the brucellosis card (BC), standard tube agglutination (STA), 2-mercaptoethanol (ME) and rivanol (RIV) tests. Antibodies were detected in the principals by day 5 and increased through day 21. The STA test was the first to become positive, followed by the BC, ME and RIV tests. After 28 days, the serologic titers receded. From day 14 through day 42, all principals had greater than or equal to 1:50 STA titers. On day 49, seven principals had greater than or equal to 1:50 STA titers and one had a 1:25 STA titer. The sentinels were negative for all tests, except sentinel number 9 which had STA titers ranging from 1:25 to 1:50 on day 14 through day 35. Blood cultures that were obtained from each principal at intervals from one hour after exposure through 49 days were negative. Brucella abortus was isolated from various lymph nodes of the eight principals and from sentinel number 9, which was apparently infected by ingesting brucellae contaminated feces from the principals. Microscopic lesions were not observed in the culture-positive tissues examined.     

Experimental infection of sheep with Brucella abortus

A case of brodifacoum poisoning is described in a six-year-old male Kelpie cross working dog. The clinical features were severe exercise intolerance, haemorrhage from the oral and nasal cavities, dyspnoea and pale mucous membranes. Diagnosis was confirmed by demonstrating an abnormally long whole blood clotting time. The dog was treated successfully by administering 1 litre of whole blood intravenously, intramuscular vitamin K₁ and a three week course of oral vitamin K₃.

Experience at the Massey University Small Animal Clinic and Hospital has indicated that poisoning of dogs with the newer long acting anticoagulant rodenticides is becoming more common.     

Pathogenesis of Brucella abortus in chicken embryos

Chicken embryos inoculated with Brucella abortus at 6, 10, and 12 days of incubation were examined by light and electron microscopy. B. abortus was identified by avidin-biotin immunoperoxidase and immunogold techniques. Death occurred from 2 to 5 days post-inoculation, depending on age of the embryo and route of inoculation. B. abortus was recovered from all infected eggs. Brucellae had spread throughout all tissues and localized preferentially within cells of mesodermal derivation. Organ distribution and degree of bacterial replication varied with age of the embryo at time of inoculation. In 6-day-old embryos, B. abortus localized preferentially in endoderm and mesoderm of yolk sac wall, extra- and intraembryonic serosal epithelia, and glomeruli of the mesonephros. In 10- and 12-day-old embryos, B. abortus spread to all tissues; renal glomeruli, liver, spleen, and heart were most severely infected. Intracellular B. abortus was within the rough endoplasmic reticulum of mesenchymal, mesothelial, yolk endodermal, and hepatic cells. In mononuclear phagocytes, endothelial cells, and granulocytes, bacteria were within membrane-bound vacuoles. Intracellular replication of B. abortus in embryonic tissues, especially yolk endoderm, closely resembled that in experimental infections of trophoblasts.     

Distribution of Brucella abortus in infected cattle

Experimentally and naturally infected cattle were examined bacteriologically to determine the anatomical distribution of specimens yielding Brucella abortus. In 91 experimentally infected pregnant cows, examined 3 to 4.5 months after conjunctival challenge during pregnancy, the most frequently infected specimen was the mammary (syn. supramammary) lymph node. All experimentally infected cows could be identified from cultures of the mammary, mandibular (syn. submaxillary), medial iliac, caudal superficial cervical (syn. prescapular) lymph nodes and uterine caruncles, cotyledons or foetal tissues. Forty-six naturally infected cows were examined and again the most frequently infected specimen was the mammary lymph node. All naturally infected cows could be identified from cultures of the mammary, parotid, mandibular and subiliac (syn. prefemoral) lymph nodes. The distribution of infected specimens was somewhat different in heifers. In 61 naturally infected heifers the most frequently infected specimen was the mandibular lymph node but 8 other specimens would have been required to enable identification of all infected heifers. Specimens from 3 infected bulls were cultured and 11 of the 12 specimens examined were infected in at least one of the bulls. The most frequently infected tissues were the mandibular, caudal superficial cervical, subiliac and scrotal lymph nodes. The results suggest which specimens should be selected for culture, particularly when only a limited amount of effort can be expended.