犬种布鲁氏菌研究进展

犬种布鲁氏菌属于6个经典种布鲁氏菌之一,为天然粗糙型,毒力较弱。长期以来,由于犬种布鲁氏菌对人类致病性较低,其危害一直被忽视。自1966年在美国发现该菌以来,已传播至世界多地。近年来,随着养犬业的不断发展及宠物犬数量的增多,犬布鲁氏菌病发病率不断上升,在某些地区已成为流行性疫病,且对人类公共卫生安全的威胁也日益加重。目前,犬种布鲁氏菌感染导致的犬布鲁氏菌病尚无可靠的诊断方法及有效的疫苗。鉴于此,在查阅相关文献的基础上,笔者对犬种布鲁氏菌的病原学、流行病学、致病机理、检测方法、疫苗研究等方面的相关研究进展进行综述,以期为犬布鲁氏菌病的深入研究和防控提供参考。     

犬布鲁氏菌菌壳疫苗的制备简

菌壳技术是一种新型的灭活疫苗制备方法,通过非变性的灭活方式保存细菌表面多个抗原表位,所制备的菌壳可作为预防细菌病的理想疫苗。本试验从噬菌体PhiX174 DNA钓取裂解E基因,连接至温控原核表达载体pBV220,通过PCR在所构建的pBV220+E上扩增出蛋白裂解部件（protein lysis component,PLC）,该部件包含阻遏蛋白cI857、溶菌E基因及终止序列rrnbT1T2,然后将其克隆至广宿主表达载体pBBR1MCS-2中,最终将构建的广宿主裂解质粒pBBR+PLC电转入犬布鲁氏菌RM6/66中。试验结果表明,经42 ℃诱导后,广宿主裂解质粒对犬布鲁氏菌RM6/66的裂解率达100%,成功制备了犬布鲁氏菌RM6/66菌壳疫苗。本试验通过菌壳技术制备的犬布鲁氏菌疫苗对预防宠物犬布鲁氏菌病起到重要作用,同时也对人兽布鲁氏菌疫苗的研制提供新策略。     

Brucella canis Isolates from Canadian Dogs

Eleven Brucella canis isolates from Canadian dogs were characterized by dye and antibiotic sensitivity, phage susceptibility, urease and H₂S production, CO₂ requirement, and reaction with monospecific A,M, and R anti-Brucella antiserum. The isolates could be separated into two distinct groups. One group had a sensitivity pattern similar to that seen with the American type strain RM666, while the other group had a pattern identical to that of a Mexican strain, Mex 51. Epidemiological studies supported contraction of infections in the United States and Mexico respectively. The characteristics of all isolates were stable after repeated subculture indicating that strain differences could serve as useful epidemiological markers and supporting division of the species into two biovars.     

Brucella canis in a HIV-infected patient

The true incidence of Brucella canis in humans is unknown, but immunocompromised patients in direct contact with dogs are thought to be at a higher risk for infection. In this case report, we present a human case of B. canis in a patient infected with human immunodeficiency virus.     

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.     

Use of phage for the identification of Brucella canis and Brucella ovis cultures

The brucella phage strains R, R/O and R/C standardised at routine test dilution on their propagating strains were effective in identifying cultures of Brucella ovis and B canis and in differentiating these from other non-smooth brucella isolates.     

Use of Brucella canis antigen for detection of ovine serum antibodies against Brucella ovis

Brucella ovis causes a genital disease of sheep manifested by epididymitis in rams and placentitis in ewes producing reduced fertility in the flock. Clinical diagnosis is not sensitive enough and bacteriological testing is not feasible for detection of the disease in large numbers of animals. Indirect methods of serological testing are preferred for routine diagnosis, of which agar gel immunodiffusion (AGID), complement fixation (CF) and ELISA tests are recommended as the most efficient. Since B. ovis shares antigenic components with Brucella canis, it would seem that either strain could be used as antigen with the same results; however, the advantage of the B. canis (M-) strain variant is that it can be used to develop a satisfactory antigen for agglutination tests. We present data on AGID and IELISA tests using B. ovis antigen and rapid screening agglutination test (RSAT), 2-mercapto-ethanol RSAT (2ME-RSAT) and IELISA using B. canis antigen. We tested 225 animals. The cut-off values were adjusted by ROC analysis using 51 negative and 32 positive sera; the IELISA-B. canis cut-off value was 39 (%P) and IELISA-B. ovis, 51 (%P), with 100% sensitivity and specificity. Of the 32 positive sera from the infected flock RSAT detected 32 (100%), 2ME-RSAT 29 (91%) and AGID 31 (97%). Of the 142 sera from suspicious flocks, 46 were negative and 56 positive in all the tests; 16 were positive by RSAT, IELISA-B. canis and IELISA-B. ovis, 20 positive only with RSAT and 2 positive only by both IELISAs. RSAT is a very sensitive screening test that, because of its simplicity and easy interpretation, following a study in larger sample, could replace AGID as a screening test for diagnosis of ovine brucellosis caused by B. ovis. The IELISA-B. canis or IELISA-B. ovis could be used as confirmatory tests, since they show equal specificity and sensitivity.     

Evaluation of immunochromatographic assay for serodiagnosis of Brucella canis

Canine brucellosis is a contagious disease with venereal and oral modes of transmission that produces late abortion in females, epididymides and prostates in males. Diagnosis is difficult because of unstable serum antibody titers that vary from individual to individual as well as between different methods used for their detection. The objective of this work was to evaluate the clinical utility of the immunochromatographic assay (ICA) for serodiagnosis of dogs suspected of having brucellosis, and results were compared with those obtained for hemoculture (HC) and the rapid screening agglutination with 2-mercaptoethanol (2-ME RSAT). The all experimentally infected dogs were positive in ICA, HC and 2-ME RSAT from 5 weeks, 7 weeks, and 3 weeks after infection, respectively. Also, among dogs selected from 10 different breed kennels occurred brucellosis, 24.8%, 39.5% and 39.1% of dogs tested were detected as positive with HC, 2-ME RSAT and ICA, respectively. The kappa value between 2-ME RSAT and ICA was 0.89. The results of this study showed that sensitivity and specificity of the ICA are comparable with those obtained using conventional serological and bacteriological test for brucellosis. In conclusion, the ICA kit provides a handy and accurate tool for the rapid serodiagnosis of canine brucellosis.     

布鲁菌分子标记菌壳株的构建与鉴定

旨在构建一种安全、高效的犬布鲁菌分子标记疫苗株。本试验将温控裂解部件(TLC)插入布鲁菌自杀质粒pBK-CMV-SacB-ΔB0419中,构建温控裂解型自杀质粒pBK-CMV-SacB-ΔB0419-TLC;通过电转化方式转入犬布鲁菌RM6/66感受态中,经卡那抗性和蔗糖培养基的正、负向筛选,将TLC片段定点插入布鲁菌B0419基因中,获得犬布鲁菌分子标记菌壳株。采用PCR法对连续30代次的菌株进行鉴定,结果显示裂解E基因和靶向插入位点区域(B0419基因)并未出现丢失和回复突变现象,表明该菌壳株具有良好的遗传稳定性。该菌壳株在培养至D_(600)值为0.6时,经42℃诱导60 h后,可获得裂解率达100%的犬布鲁菌菌壳;经超薄切片和染色处理后,在透射电镜观察下可见形态完整的空心结构,其内容物明显减少。本试验结合细菌菌壳技术与同源重组技术,成功构建了具有分子标记特征的犬布鲁菌菌壳株,为新型布鲁菌疫苗的研制提供策略。     

Genetic comparison of Brucella canis isolates by the MLVA assay in South Korea

The multiple-locus VNTR analysis (MLVA) assay is a method frequently employed as a molecular epidemiological tool for Brucella genetic fingerprinting. The purpose of this study was to assess the genotyping of 77 B. canis isolates from 14 different dog breeding farms in Korea by the MLVA assay and to compare the epidemiological relationships between the Korean isolates and foreign ones. Simpson's diversity index for 17 loci showed a range from 0 to 0.846 in 77 B. canis isolates. B. canis isolates in Korea were observed to have high genetic diversity at the most variable loci and were divided into 30 distinct genotypes by phylogenetic analysis. Some B. canis isolates were closely related to previously typed isolates in other countries. The MLVA assay can be helpful to analyze the epidemiological correlation of B. canis isolates in domestic pet animals and to track the geographic origin by comparing the genetic patterns with foreign isolates. Therefore, the MLVA assay will be useful as a tool for control and preventive measures of canine brucellosis.     

Peripheral lymphocyte function in dogs with Brucella canis infection

Peripheral lymphocyte function in dogs with Brucella canis infection was evaluated using in vitro lymphocyte stimulation with the mitogens PHA, Con A and PWM, and killed Brucella canis organisms. Bitches with naturally occurring Brucella canis infection were compared to negative controls. There was no difference in the response to Con A and PWM between these two groups. Lymphocytes from infected dogs were less responsive (p less than .05) to PHA than were lymphocytes from controls. There was a significant (p less than .005) difference in response to Brucella canis antigen between the two groups. Lymphocytes from infected dogs were stimulated by Brucella canis antigen, whereas those from controls did not respond.