A study of cattle infected with Brucella abortus and which showed aberrant serological reactions

SUMMARY Sixty cows, 48 of which had been vaccinated with live Brucella abortus strain 19 (S19) or with killed B. abortus strain 45/20 (S45/20) and 12 of which were unvaccinated animals, were challenged with B. abortus strain 544. Ten of the 27 cattle found to be infected after challenge showed aberrant serological reactions to the Rose Bengal Plate test, serum agglutination test and/or complement fixation test. These 10 cattle were all previously vaccinated with S19 or S45/20. It was concluded that infection in cattle vaccinated with S19 or S45/20 may be more difficult to detect than infection in animals that have no history of vaccination.     

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.     

The Dutch Brucella abortus monitoring programme for cattle: the impact of false-positive serological reactions and comparison of serological tests

The Dutch national Brucella abortus eradication programme for cattle started in 1959. Sporadic cases occurred yearly until 1995; the last infected herd was culled in 1996. In August 1999 the Netherlands was declared officially free of bovine brucellosis by the European Union. Before 1999, the programme to monitor the official Brucella-free status of bovine herds was primarily based on periodical testing of dairy herds with the milk ring test (MRT) and serological testing of all animals older than 1 year of age from non-dairy herds, using the micro-agglutination test (MAT) as screening test. In addition, serum samples of cattle that aborted were tested with the MAT. The high number of false positive reactions in both tests and the serum agglutination test (SAT) and complement fixation test (CFT) used for confirmation seemed to result in unnecessary blockade of herds, subsequent testing and slaughter of animals. For this reason, a validation study was performed in which three indirect enzyme-linked immunosorbent assays (ELISAs), the CFT and the SAT were compared using a panel of sera from brucellosis-free cattle, sera from experimentally infected cattle, and sera from cattle experimentally infected with bacteria which are known to induce cross-reactive antibodies (Pasteurella, Salmonella, Yersinia, and Escherichia). Moreover, four ELISAs and the MRT were compared using a panel of 1000 bulk milk samples from Brucella-free herds and 12 milk samples from Brucella abortus- infected cattle. It is concluded that the ELISA obtained from ID-Lelystad is the most suitable test to monitor the brucelosis free status of herds because it gives rise to fewer false-positive reactions than the SAT.     

Comparison of an enzyme-linked immunosorbent assay using monoclonal antibodies and a complement fixation test for cattle vaccinated and infected with Brucella abortus

A competitive enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody conjugated to horseradish peroxidase MA(A) and a complement fixation test (CFT) were applied to sera collected over a two-year period from 60 cattle challenged with Brucella abortus strain 544. Forty-eight of the cattle were previously vaccinated with B. abortus strain 19 (S19) or B. abortus strain 45/20 (45/20). After challenge 33 of the cattle remained uninfected and nine of the 27 infected cattle showed aberrant reactions by the CFT. The performance of the MA(A) ELISA was as follows: after vaccination, the MA(A) ELISA, like the CFT, was unable to differentiate infected cattle from those recently vaccinated with S19. After challenge the MA(A) ELISA gave results comparable with the CFT for those cattle with aberrant reactions. For the non-infected cattle there was a similar number of weeks after challenge when both tests were negative. It is suggested that the main advantage of the MA(A) ELISA when compared with the CFT lies in its relatively simple test procedure.     

Cell-mediated immune responses in cattle adult-vaccinated with Brucella abortus strain 19 and in cattle infected with Brucella abortus field strain.

Cell-mediated immune responses in cattle adult-vaccinated with Brucella abortus strain 19, cattle infected with B abortus field strain, and nonexposed cattle were studied by an in vitro lumphocyte-stimulation test (LST). Lymphocytes were prepared from peripheral bovine blood by the Ficoll-diatrizoate technique, and results were assayed for [3H]thymidine incorporation into DNA by liquid scintillation spectrometry. Serotests and bacteriologic isolation attempts were conducted simultaneously with LST. Lymphocytes from cattle infected with field strains had significantly (P = 0.01) higher specific lymphocyte-stimulation inexposed controls. The LST, the serum standard-tube agglutination test (STT), the Rivanol (RIV) test, and the complement-fixation (CF) test correctly classified cattle from which field strains and strain 19 of B abortus were isolated. The LST was negative in cattle vaccinated with B abortus strain 19 (nonshedding), but the three serotests had many false-positive reactions. The CF test had the least false-positive reaction, followed by the RIV test, and the STT was the least specific. Well before the three serotests became positive, the LST was positive in samples from some cattle during the incubation period of the infection. There was little or no correlation between cell-mediated immune responses (as measured by LST) and serum antibody responses (as measured by STT, RIV test, and CF test) in vaccinated but culture-negative cattle and in some nonvaccinated cattle during the incubation period.     

Specific lymphocyte stimulation in cattle naturally infected with strains of Brucella abortus and cattle vaccinated with Brucella abortus strain 19.

Cell-mediated immune responses in cattle naturally infected with strains of Brucella abortus and in cattle vaccinated with B abortus strain 19 during calfhood were studied by an in vitro lymphocyte-stimulation procedure. Lymphocytes were prepared from peripheral bovine blood by the Ficoll-diatrizoate technique, suspended in RPMI-1640 medium (1.5 X 10(6) lymphocytes/ml), cultured with B abortus-soluble antigen or phytohemagglutinin, and incubated for 6 days. Sixteen hours prior to termination of incubation, cultures were labeled with 1 muCi of [3H]thymidine (3HdT) and, after harvesting, assayed for 3HdT incorporation into DNA by liquid scintillation spectrometry. Lymphocytes from cattle with bacteriologically confirmed isolation of B abortus underwent a significantly higher lymphocyte stimulation with B abortus-soluble antigen than did cattle vaccinated with B abortus strain 19 during calfhood (P less than 0.005). Standard seroagglutination tests were conducted simultaneously with lymphocyte-stimulation tests, but there was no apparent correlation between levels of humoral antibodies and the cell-mediated immune responses as measured by in vitro specific lymphocyte stimulation.     

The relationship between the isolation of Brucella abortus and serological status of infected, non-vaccinated cattle

Seventy two non-vaccinated cattle with various complement fixation (CF), rose bengal (RB) and enzyme-linked immunosorbent assay (ELISA) results at slaughter were examined bacteriologically and serologically. Brucella abortus was recovered from 49 (68.1%) of the cattle and the use of a biphasic culture medium was entirely responsible for the detection of 6 (12.2%) of the culture positive cattle. The supramammary and retropharyngeal lymph nodes were the most rewarding tissues to culture. A comparison of culture results and serological status demonstrated that B. abortus could be isolated from cattle with negative RB and CF tests and that the ELISA was useful in detecting these cattle and infected cattle with low CT titres. The RB test was also useful as it detected all but 4 of the cattle found to be infected.     

Infection of cattle in Kenya with Brucella abortus biovar 3 and Brucella melitensis biovar 1 genotypes

Brucella melitensis biovar 1 was isolated from bovine milk samples from a herd in central Kenya, and Brucella abortus biovar 3 was isolated from aborted fetus materials and vaginal discharge fluids from cattle in central and eastern provinces of Kenya. All infections including those with B. melitensis were in cattle with reproductive problems kept in mixed herds indicating that cross infection occurs from small ruminants. Multiple-locus variable-number tandem repeat analysis genotyping revealed a close molecular homology of the B. melitensis isolates with an isolate from Israel and a close homology of the B. abortus isolates with an isolate from Uganda indicating that these genotypes have a wide geographic distribution. Infection of cattle with B. melitensis may complicate the control of brucellosis in this country.     

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.     

Biotypes of Brucella abortus and their value in epidemiologic studies of infected cattle herds.

Four Texas cattle herds containing cows infected with either Brucella abortus biotype 1, 2, or 4 were studied to determine the probability of transmission of Brucella between adjacent cattle herds, the most probable means by which Brucella was introduced into the herds, and the relative frequency of strain 19 isolation from vaccinated cattle. A total of 1,935 cattle in the four herds were tested for brucellosis; 339 reactors were identified, and isolations of B abortus were made from 143. The biotype of B abortus was used to determine that purchased cattle or reentry of bred heifers into the herds was probably responsible for introducing B abortus and that the biotype was not readily transmitted to adjacent herds. Three (9%) of 32 B abortus isolations from adult-vaccinated cattle were strain 19. The data supported the hypothesis that biotypes can be useful in determining the source of B abortus for cattle and in differentiating field and vaccine strain infections in adult-vaccinated cattle.     

Enhanced efficacy of recombinant Brucella abortus RB51 vaccines against B. melitensis infection in mice

Brucella abortus strain RB51 is an attenuated rough strain, currently being used as the official live vaccine for bovine brucellosis in the USA and several other countries. In strain RB51, the wboA gene, encoding a glycosyltransferase required for the O-side chain synthesis, is disrupted by an IS711 element. Recently, we have demonstrated that strain RB51WboA, RB51 complemented with a functional wboA gene, remains rough but expresses low quantities of O-side chain in the cytoplasm. Mice vaccinated with strain RB51WboA develop greatly enhanced resistance against challenge with B. abortus virulent strain 2308. We have also demonstrated that overexpression of Cu/Zn superoxide dismutase (SOD) in strain RB51 (RB51SOD) significantly increases its vaccine efficacy against strain 2308 challenge. In this study, we constructed a new recombinant strain, RB51SOD/WboA, that over expresses SOD with simultaneous expression of O-side chain in the cytoplasm. We tested the vaccine potential of strains RB51SOD, RB51WboA, RB51SOD/WboA against challenge with virulent Brucella melitensis 16M and B. abortus 2308 in mice. In comparison with strain RB51, strain RB51SOD induced better protection against strain 2308, but not strain 16M, challenge. Similar to strain RB51WboA, vaccination with strain RB51SOD/WboA resulted in complete protection of the mice from infection with strain 2308. When challenged with strain 16M, mice vaccinated with either strain RB51WboA or strain RB51SOD/WboA were significantly better protected than those vaccinated with strain RB51 or RB51SOD. These results suggest that strains RB51WboA and RB51SOD/WboA are good vaccine candidates for inducing enhanced protection against B. melitensis infection.     

Identification of Brucella abortus, B. canis, B. melitensis, and B. suis by carbon substrate assimilation tests

By using the results of seven carbon substrate assimilation tests from the Biotype 100 system (bioMérieux, Marcy-l’Etoile, France), we correctly identified 79 (85.9%) of 92 Brucella strains tested. The specificity of the method varied from 97.4 to 100% depending on the species. Although a biological safety cabinet must be used, this method represents an easy and fast alternative for the identification of Brucella species.     

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。