Molecular evidence of new variant Brucella in North Pacific common minke whales

Brucella, a causative agent of brucellosis, has been isolated recently from a variety of marine mammals. The molecular analysis of marine mammalian Brucella strains, without manifest pathology of brucellosis in the eastern North Atlantic, showed that they are distinct from terrestrial Brucella species. Previously, we reported abnormal gonads in common minke whales (Balaenoptera acutorostrata) in the western North Pacific and suggested the presence of Brucella infection in the whales in pathology and serology studies. In the present study, using polymerase chain reaction (PCR), Brucella was detected in granular testes of the whales showing caseation or calcification. The insertion of an IS711 transposable element specific for marine mammal isolates as well as a seal isolate-specific DNA fragment were also found. Molecular characterization of Brucella based on sequence analysis of the PCR products amplified from the outer membrane protein (omp) 2 gene showed that the Brucella from North Pacific common minke whales was different from terrestrial and North Atlantic marine mammal Brucella strains. The North Pacific Brucella showed the highest similarity to North Atlantic seal strains among the known Brucella strains.     

Application of biotechnology in the diagnosis and control of brucellosis in the Near East Region

Although much information is available on the diagnosis and control of brucellosis, most of it is concerned with Brucella abortus in cattle. In contrast, no specific serological test for Brucella melitensis infection in small ruminants has been developed. Antigens prepared from Brucella abortus, as well as tests used for diagnosis of Brucella abortus infection in cattle, are also used for diagnosis of Brucella melitensis in small ruminants, buffaloes, camels, swine and other animals. Control policy for Brucella melitensis has not yet been established in all countries of the region, probably due to lack of information on the best methods. Moreover, little information exists on the efficacy of a mass vaccination strategy in small ruminants, regarding vaccine strain persistence in the host and its excretion in the milk of adult animals, or horizontal transfer in the field. In addition, recent data have suggested that Brucella melitensis may have evolved to more virulent strains which represent a higher risk for humans. It is suggested that a project should be financed to establish or develop a feasible regional brucellosis control programme, whose objectives will be: (i) to survey different animal species and evaluate the applicability of several serological and molecular biological techniques using specific diagnostics prepared by genetic engineering; (ii) to characterize bacteriological and genetic properties of Brucella isolates as well as the Brucella melitensis Rev. 1 vaccinal isolates recovered from different animals in various countries of the region and compare them to the standard Elberg strain. This will provide an insight into the mechanisms involved in the attenuation of the strain and its immune properties. This knowledge will be used to boost the immune protection conferred by conventional Brucella melitensis Rev. 1 vaccination, to develop new effective attenuated vaccine and/or to design subunit or DNA vaccines for whole flock vaccination.     

Temporal analysis of pathogenic events in virulent and avirulent Brucella melitensis infections

Despite progress in mouse models of brucellosis, much remains unknown regarding Brucella dissemination and tissue localization. Here, we report the dynamics of Brucella infection in individual mice using bioluminescent Brucella melitensis. Bioluminescent imaging of infected interferon regulatory factor-1 knockout (IRF-1(-/-)) mice identified acute infection in many tissues. Brucella was found to replicate in the salivary glands of IRF-1(-/-) and wild-type C57BL/6 mice suggesting a previously unknown tissue preference. Establishing a niche in this region may have relevance in humans where infection can result from ingestion of few bacteria. Sublethal infection of IRF-1(-/-) mice resulted in chronic Brucella localization in tail joints, an infection parallel to osteoarticular brucellosis in humans. Importantly, bioluminescent imaging rapidly identified attenuated EZ::TN/lux mutants in infected mice and revealed differences in dissemination, thereby defining the contribution of Brucella genes to virulence and tissue localization. Surprisingly, a virB mutant, though defective in persistence, disseminated similarly to virulent Brucella, suggesting bacterial spread is independent of VirB proteins that are important for intracellular survival. Together, our results reveal kinetics of acute and chronic Brucella infection in individual mice that parallels human infection as well as readily identified attenuated bacteria. Our approach facilitates identifying virulence determinants that may control tissue specific replication and may help develop therapeutics to overcome Brucella-induced chronic debilitating conditions.     

Comparison of three different PCR methods for detection of Brucella spp in human blood samples

In the diagnosis of human brucellosis, PCR could be a more sensitive technique than blood cultures and more specific than conventional serological tests. We compared three different PCR methods for the detection of Brucella spp. and we studied whether human genomic DNA affect the sensitivity of three primer pairs for the detection of Brucella DNA in a peripheral-blood PCR assay. These three pairs of primers amplified three different fragments included in: (i). a gene encoding a 31-kDa Brucella abortus antigen (primers B4/B5), (ii). a sequence 16S rRNA of B. abortus (primers F4/R2), and (iii). a gene encoding an outer membrane protein (omp-2) (primers JPF/JPR). The three primers assayed showed a difference in sensitivity for detecting purified Brucella DNA, ranging between 8 fg and 20 pg. However, the sensitivity of the primers F4/R2 and B4/B5 was affected by the presence of human DNA while the primers JPF/JPR were not. Therefore, although the sensitivity of PCR using primers F4/R2 is affected by human DNA, they are still the most sensitive and they could provide a useful tool for the diagnosis of human brucellosis.     

Brucella abortus in wildlife on selected cattle farms in Alabama

Two studies of brucellosis in wildlife on farms where the brucellosis infection prevalence in cattle was known are reported. On a research farm, 233 feral animals of 22 mammalian species and 12 of seven avian species were trapped during three time periods. Sixty were studied before cattle were introduced, 128 were studied while 501 cattle infected with Brucella abortus were calving and aborting, and 60 specimens were collected 20 mo after the last infected cow calved. Selected tissues from 229 wild animals were cultured and sera from 138 were examined using the brucellosis card, standard tube agglutination (STA), 2-mercaptoethanol (2-ME) and rivanol (RIV) tests. Brucella abortus was not recovered from any animals sampled prior to cattle being introduced and all sera collected were negative. Brucella abortus was isolated from four opossums (Didelphis virginiana) and one raccoon (Procyon lotor) in the group of animals trapped during the calving period. Three serums were tested and had STA titers ranging from 1:100 to 1:200. Of 68 sera only one had antibodies. Brucella were not isolated from 59 animals trapped after the calving period and only one of 42 serums had antibodies. On regional cattle farms, 243 wild animals were trapped. Brucellae were not isolated from 223 animals which were cultured. No serums had significant titers. The data from this study suggest opossums and raccoons can be infected from cattle but are unlikely to maintain the infection.     

上海地区散发布氏杆菌感染的细菌学及分子鉴定

目的 本研究对我院的1例散发布氏杆菌病患者进行细菌学及分子生物学的分析,并在国内首次尝试了用数目可变串联重复单元(VNTR)分子指纹分析法对其进行了基因分型并和国际流行株进行了分子流行病学比较分析。方法 对临床疑似布氏杆菌病病例作血液细菌培养与生化鉴定,进一步作布氏杆菌特异性基因片段的序列分析鉴定以及利用布氏杆菌基因组中的8个位点构建VNTR指纹图谱,参照国际布氏杆菌VNTR数据库,构建布氏杆菌基因系统树。结果 用细菌学方法确定散发疑似布氏杆菌病病例体内分离到的为布氏杆菌,通过基因序列分析进一步得到证实,但不能鉴定到生物种和生物型。对分离株作VNTR指纹分析提示该散发布氏杆菌病为猪2型布氏杆菌感染所致。结论 通过传统细菌培养方法与布氏杆菌VNTR指纹分析可用于我国布氏杆菌病分子流行病学的系统调查。     

Evaluation of Brucella MLVA typing for human brucellosis

Human brucellosis is still the most common bacterial zoonosis worldwide. Neither well-known molecular tools nor the classical biotyping methods are satisfactory for subtyping of Brucella spp. Loci containing Variable Number of Tandem Repeats (VNTRs) have recently proved their usefulness in typing strains from animal origin despite the high genetic homogeneity within the genus Brucella (DNA-DNA homology >90%). The aim of this study was to evaluate MLVA (Multiple Locus VNTR Analysis) for diagnostic and epidemiological use in human brucellosis. One hundred and twenty-eight B. melitensis isolates of all three biovars were typed using eight minisatellite (panel 1) and eight microsatellite (panel 2) markers. One hundred and ten different genotypes were identified. The MLVA clustering pattern correlated with the geographic origin of the strains. Brucella strains isolated from different patients within the same outbreak or from the same patient before first-line therapy and after relapse showed identical genotypes. Fuchsin sensitive B. melitensis strains were found in closely related clusters giving evidence for an association between VNTRs and some phenotypic characteristics. However, the validity of biovars established by classical microbiological methods could not be confirmed by MLVA clustering. The original data can be queried on the genotyping web page at http://bacterial-genotyping.igmors.u-psud.fr. The MLVA assay is rapid, highly discriminatory, and reproducible within human Brucella isolates. MLVA can significantly contribute to epidemiological trace-back analysis of Brucella infections and may advance surveillance and control of human brucellosis.     

Comparison of different PCR methods for detection of Brucella spp. in human blood samples

For detection of Brucella species by PCR four DNA extraction methods and four targets were compared using pure culture of Brucella melitensis and the best conditions were applied in clinical samples. It was found that the MagNA Pure LC method was the most efficient and sensitive method showing a positive PCR reaction with DNA extracted from as low as 25 and 100 CFU suspended in one ml blood and one ml water, respectively. Detection of Brucella spp. by conventional PCR was investigated using four different targets. The results indicated that The B4-B5 amplification method was the most sensitive one as it could amplify DNA extracted from as a low as 25 and 100 CFU/ml suspended in one ml water and blood, respectively. Furthermore real-time PCR was able to detect Brucella using DNA extracted from as low as 50 CFU/ml blood and 15 CFU/ml water, respectively. The best and optimum detection conditions were applied to the clinical samples. Evaluation of conventional PCR assays on blood specimens confirmed 72% of the results obtained by conventional blood culture methods with a specificity of 95%, while serum samples had a sensitivity of 54% and specificity of 100%. Real-time PCR was generally found to be more sensitive and specific for detecting Brucella spp. in blood and serum samples compared to conventional PCR. The real-time PCR done on blood specimens confirmed 77.5% of the results obtained by conventional blood culture methods with specificity of 100%, while 60% of serum samples were found to be positive with specificity of 100%. These results suggest that serum and blood analysis by conventional and real time PCR is a convenient and safe method for rapid and accurate diagnosis of brucellosis.     

Comparative evaluation of recombinant BP26 protein for serological diagnosis of Brucella melitensis infection in goats

An enzyme-linked immunosorbent assay (ELISA) was developed for the serological diagnosis of caprine brucellosis with purified recombinant BP26 (outer membrane protein 28) of Brucella melitensis 16M produced in Escherichia coli. Brucella protein named CP28, BP26, or Omp28 has been identified as an immunodominant antigen in infected cattle, sheep, goats, and humans. The recombinant BP26 (rBP6) ELISA performed with serum samples (n = 1738) taken from organized farms and field goats from Northern India and tested in two different batches of 778 and 960 animals for brucellosis. Positive results were compared with the traditional serum agglutination test (SAT), complement fixation test (CFT) and dot-ELISA. The diagnostic sensitivity of rBP26 ELISA, SAT, CFT and dot-ELISA was 87.5%, 56.25%, 62.5% and 75% respectively. The specificity of the rBP26 ELISA, SAT, CFT and dot-ELISA was 90%, 75%, 80%, and 85% respectively. The results of rBP26 ELISA positive animals were further compared and evaluated by tissue PCR using B. melitensis BP26 gene as target. This resulted in 100% positive correlation between rBP26 ELISA and BP26 PCR. Thus, these results confirmed the importance of BP26 as a suitable antigen and rBP26 ELISA, if well standardized, proved to be a good screening test for the serological diagnosis of caprine brucellosis.     

Brucella suis infections in collared peccaries in Venezuela

A bacteriologic and serologic study was conducted on two ranches in the states of Apure and Guarico, Venezuela for brucellosis in collared peccaries (Tayassu tajacu). One hundred thirty-nine peccaries were necropsied and tissues were cultured. Forty-three isolations of Brucella suis biovar 1, were made from lymph nodes and spleens of 25 males and 18 females. Antibody to Brucella sp. was detected in sera from 122 animals by the rapid plate agglutination, standard tube agglutination, 2-mer-captoethanol, rivanol, complement fixation and card tests. Young animals had infection and reactor rates nearly as high as the older animals indicating most were infected at a relative early age. Results suggest that this species may transmit brucellosis when living with domestic animals. This is the first report of B. suis biovar 1 from collared peccaries in Venezuela.     

Differentiation of Brucella species by random amplified polymorphic DNA analysis

Random amplification of polymorphic DNA (RAPD) was used for discrimination between 46 Brucella strains and 14 representatives of the alpha-2 and alpha-1 subgroups of Proteobacteria. To evaluate a relatively quick and exact method for Brucella identification, the authors specified the most suitable conditions for RAPD amplification of Brucella DNA with two 10-mer primers, containing lower and higher percentages of G and C. The software package PHYLIP 3.1 was used for cluster analysis of the RAPD fingerprints. The optimization of RAPD conditions resulted in PCR mixes suitable for reliable typing of Brucellae. The distance-based methods (Fitch-Margoliash, UPGMA and Neighbour-joining) gave clear discrimination between Brucella species. The constructed dendrograms put Br. canis and Br. suis bv. 1 in the same cluster and differentiated Brucella strains according to their host preferences. RAPD can be useful method to distinguish related bacterial species, and under strictly established conditions the reaction appears to be a simple, quick and sensitive technique for the epidemiological investigation of brucellosis.     

Heterogeneous shedding of Brucella abortus in milk and its effect on the control of animal brucellosis

Aims: To ascertain whether in Brucella abortus‐infected water buffalo herds, the number of newly infected animals could be reduced by culling superspreaders (the animals secreting ≥10⁴ CFU per ml of milk). Methods and Results: The number of B. abortus present in the milk (CFU per ml) from 500 water buffaloes was measured by the culture. Each animal was tested three times, at one month intervals. The presence or the absence of B. abortus in each milk sample was confirmed by PCR. A majority of infected animals shed the pathogen at a low level (≤10³ CFU ml^?1). However, a few infected individuals (superspreaders) shed large numbers of B. abortus (≥10⁴ CFU ml^?1). Quantitative PCR of B. abortus positive milk samples gave comparable results to culture. Culling of the superspreaders was sufficient to arrest the spread of infection. Significance and Impact of the Study: The approach described here can reduce significantly the cost of controlling brucellosis. Culture and quantitative PCR tests identify superspreaders and, compared with the serological tests in use to detect brucellosis, provide also a more accurate estimate of the disease incidence.     

A comparison of four serologic assays in screening for Brucella exposure in Hawaiian monk seals

A survey for Brucella spp. antibodies was undertaken on 164 serum samples from 144 Hawaiian monk seals (Monachus schauinslandi) from the northwestern Hawaiian Islands collected between 1995 and 2002. The buffered antigen plate agglutination test (BPAT), the indirect enzyme immunoassay (I-ELISA), the competitive enzyme immunoassay (C-ELISA), and the fluorescence polarization assay (FPA) were compared with regard to their ability in detecting antibodies to Brucella spp. in the serum samples. Overall, antibodies were detected in 28 (17.1%) animals, using the BPAT test, 25 (15.2%) by the C-ELISA, and 19 (11.6%) in the I-ELISA and the FPA test, using thresholds established for cattle. No evidence of gross pathology consistent with clinical brucellosis was noted in any of the seropositive animals tested. Although further work would be necessary to validate these tests for use with monk seals it appears that both the C-ELISA and the FPA tests would be appropriate as diagnostic screening tests for detection of antibodies to Brucella spp. in this species.     

Comparison of culture and PCR for the detection of Brucella melitensis in blood and lymphoid tissues of serologically positive and negative slaughtered sheep

Aims: To compare the culture and PCR methods for detection of Brucella melitensis in blood and lymphoid tissue samples obtained from slaughtered sheep (n = 162) testing positive/negative in serological tests (Rose Bengal test and serum agglutination test). Methods and Results: Of 162 sheep examined, 45 were positive and 117 negative in serological tests. A PCR assay based on a pair of Br. melitensis‐specific primers was used to detect DNA in blood and lymphoid tissue. Brucella melitensis was isolated from 1·2% (2/162) and 17·2% (28/162) of the blood and lymphoid tissue samples respectively. Positive PCR products with a molecular size of 731 bp were obtained from 27·7% (45/162) of blood and 29·0% (47/162) of lymphoid tissue samples. Conclusions: The species‐specific PCR assay detected a higher number of Br. melitensis DNA both from serologically positive (P < 0·01 in blood PCR, P < 0·001 in tissue PCR) and serologically negative (P < 0·001 in both blood PCR and tissue PCR) sheep compared with classical bacteriological culture methods. Significance and Impact of the Study: The results emphasize the importance of using more than one type of diagnostic technique for the detection of animals positive for brucellosis, especially with epidemiological purposes.     

Analysis of PCR-diagnosis of brucellosis in human]

Polymerase chain reaction (PCR) was used for the diagnosis of brucellosis in humans with different forms of this disease. A high incidence (77.6%) of Brucella infection was revealed in the staff of cattle breeding centers with unfavorable situation with regard to brucellosis. Such a conclusion was made after PCR testing of native human sera. In acute brucellosis of humans amplification of the specific site of brucella DNA in PCR is possible only after extraction of DNA by a procedure adapted for DNA extraction from intact brucella cells. In chronic infection weak amplification of brucella genome DNA fragment was observed in investigation of native sera by the PCR. More expressed amplification product was recorded in PCR with a DNA precipitate from this serum obtained by ethanol precipitation. A still higher level of brucella DNA fragment amplification was observed after DNA extraction from sediment obtained by ethanol precipitation from this serum. These data confirmed the incomplete phagocytosis phenomenon at the early stage of infection, known in brucellosis pathogenesis, and allowed some hypotheses on the pathogenesis of chronic phase of brucellosis infection.     

Comparative proteome analysis of Brucella melitensis vaccine strain Rev 1 and a virulent strain, 16M

The genus Brucella consists of bacterial pathogens that cause brucellosis, a major zoonotic disease characterized by undulant fever and neurological disorders in humans. Among the different Brucella species, Brucella melitensis is considered the most virulent. Despite successful use in animals, the vaccine strains remain infectious for humans. To understand the mechanism of virulence in B. melitensis, the proteome of vaccine strain Rev 1 was analyzed by two-dimensional gel electrophoresis and compared to that of virulent strain 16M. The two strains were grown under identical laboratory conditions. Computer-assisted analysis of the two B. melitensis proteomes revealed proteins expressed in either 16M or Rev 1, as well as up- or down-regulation of proteins specific for each of these strains. These proteins were identified by peptide mass fingerprinting. It was found that certain metabolic pathways may be deregulated in Rev 1. Expression of an immunogenic 31-kDa outer membrane protein, proteins utilized for iron acquisition, and those that play a role in sugar binding, lipid degradation, and amino acid binding was altered in Rev 1.     

Preliminary development of a diagnostic test for Brucella using polymerase chain reaction

A highly sensitive and specific diagnostic test for Brucella based on polymerase chain reaction is under development in our laboratory. A commercially available PCR kit was used to create primers that allowed the amplification of a 635 bp fragment of a 43 kDa outer membrane protein gene from Brucella abortus strain 19. We successfully amplified the cloned gene present in the pMS64 plasmid and genomic Brucella S19 DNA. The amplified DNA was easily detected by agarose gel electrophoresis. Using both the pMS64 plasmid and Br. abortus S19 purified DNA as template each component of the PCR reaction was adjusted for the optimum amplification of the DNA sequence. Optimum specific amplification resulted when the primer annealing temperature was 60C. The gene fragment was amplifiable in 25 different Brucella species and strains. To test the specificity of the reaction, DNA extracted from 17 micro-organisms possibly associated with cattle were tested. No amplification was observed. The sensitivity of the reaction was determined with different concentrations of genomic Brucella strain 19 DNA. As little as 0.1 pg DNA (less than 100 brucella cells) could be detected. The specificity and sensitivity of PCR combined with its simplicity and speed suggests the potential of this technique for routine diagnosis of brucellosis.     

Preliminary development of a diagnostic test for Brucella using polymerase chain reaction

A highly sensitive and specific diagnostic test for Brucella based on polymerase chain reaction is under development in our laboratory. A commercially available PCR kit was used to create primers that allowed the amplification of a 635 bp fragment of a 43 kDa outer membrane protein gene from Brucella abortus strain 19. We successfully amplified the cloned gene present in the pMS64 plasmid and genomic Brucella S19 DNA. The amplified DNA was easily detected by agarose gel electrophoresis. Using both the pMS64 plasmid and Br. abortus S19 purified DNA as template each component of the PCR reaction was adjusted for the optimum amplification of the DNA sequence. Optimum specific amplification resulted when the primer annealing temperature was 60 degrees C. The gene fragment was amplifiable in 25 different Brucella species and strains. To test the specificity of the reaction, DNA extracted from 17 micro-organisms possibly associated with cattle were tested. No amplification was observed. The sensitivity of the reaction was determined with different concentrations of genomic Brucella strain 19 DNA. As little as 0.1 pg DNA (less than 100 brucella cells) could be detected. The specificity and sensitivity of PCR combined with its simplicity and speed suggests the potential of this technique for routine diagnosis of brucellosis.     

犬布鲁氏菌病

目前,犬布鲁氏菌病在全球多个国家和地区都有发生,而且发病数量逐年上升,由犬布鲁氏菌病感染人的病例报道也日益增多。因此,犬布鲁氏菌病的控制和根除对人类健康和公共卫生安全具有重要的意义。据报道,我国多个省份存在犬布鲁氏菌病,而且感染和发病率有上升的趋势。由于我国养犬数量巨大,犬与人类接触密切,病犬作为人类布鲁氏菌病的传染源不容忽视。犬布鲁氏菌病的研究起步较晚,流行病学资料欠完善,公共卫生学意义有待阐明,诊断、免疫预防等诸多问题还没完全解决,加强犬布鲁氏菌病的研究和防控应引起足够的重视。本文就犬布鲁氏菌病的病原学、流行病学、病理学、临床症状、诊断、治疗、预防及公共卫生安全等分别做一阐述。