Diagnosis of canine brucellosis by ELISA using an antigen obtained from wild Brucella canis

An indirect ELISA test was developed for the diagnosis of Brucella canis infection in dogs. A bacterial whole cell extract was used as a solid phase antigen, using B. canis isolated from an infected animal. Sera from culture-positive and healthy negative animals were used as internal reference controls. The cut-off point was determined by a mathematical formula for a statistically valid value, which defined the upper prediction limit, based on the upper tail of the t-distribution of 21 negative control sera readings, for the confidence level of 99.5%. The sensitivity and specificity of the ELISA test were 95% and 91%, respectively. The ELISA test showed a significant concordance index (K=0.84) with the agar gel immunodiffusion test. The reliability of the ELISA for the detection of infected animals was established by a double blind study testing 280 sera provided by serum banks from different diagnostic and research institutions and analyzed by ROC Curve.     

Diagnosis of canine brucellosis by ELISA using an antigen obtained from wild Brucella canis

An indirect ELISA test was developed for the diagnosis of Brucella canis infection in dogs. A bacterial whole cell extract was used as a solid phase antigen, using B. canis isolated from an infected animal. Sera from culture-positive and healthy negative animals were used as internal reference controls. The cut-off point was determined by a mathematical formula for a statistically valid value, which defined the upper prediction limit, based on the upper tail of the t-distribution of 21 negative control sera readings, for the confidence level of 99.5%. The sensitivity and specificity of the ELISA test were 95% and 91%, respectively. The ELISA test showed a significant concordance index (K = 0.84) with the agar gel immunodiffusion test. The reliability of the ELISA for the detection of infected animals was established by a double blind study testing 280 sera provided by serum banks from different diagnostic and research institutions and analyzed by ROC Curve.     

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.     

一例犬吉氏巴贝斯虫病的诊治

3岁雄性贵宾犬,体重3.20 kg,免疫完全,未绝育,从未进行过体内外驱虫,院内散养。主诉最近2周发现犬的精神状态渐差,进食量逐渐减少。为了对其进行诊治,采用体格检查、血液学检查以及影像学检查的方法进行诊断,并根据诊断结果进行治疗,结果表明,该犬感染吉氏巴贝斯虫且并发肝脏损伤,通过采取输血、驱虫等治疗措施,患犬的临床症状消失,镜检外周血虫体消失,治疗后未见复发。说明临床上可通过血液学检测对犬的吉氏巴贝斯虫感染进行确诊,且输血结合驱虫治疗可有效治疗该病。     

Transmission of Brucella canis by contact exposure

Transmission studies demonstrated that canine brucellosis can spread from infected to susceptible males maintained in close contact after 4 to 6 months of cohabitation. Spread by males occurred after epididymitis was observed in the infected dogs. Transmission via contaminated urine was suspected, but not proved. The bladder urine of infected males, probably contaminated with seminal fluid, contained higher numbers of B. canis organisms than did that of female dogs. Highest concentrations of bacteria in urine were found between postinfection weeks 8 and 12. Infected females transmitted the infection to contact females after 5 months. Immature females or males infected with B. canis did not transmit brucellosis until after an estrus or a mating was observed--about post-contact months 10-12.     

钩端螺旋体引起犬肾功能衰竭的诊断与治疗

为判定肾功能衰竭病原及引起的相关症状,并提供合理的诊治方案,本研究对一例柯基病犬进行表征、血常规、血液生化、血气及病原进行检测。结果显示,与正常参考值相比,病犬前期血常规检测中淋巴细胞比例（W-SCR）、中间细胞比例（W-MID）、中间细胞（MID）及红细胞分布宽度（RDW-CV）升高;粒细胞比例（W-LCR）、红细胞压积（PCV）、红细胞平均体积（MCV）、血红蛋白含量（MCH）、红细胞分布宽度（RDW-SD）明显降低。与正常参考值相比,生化指标中钙离子（Ca²⁺）、血糖（GLU）、尿素氮（BUN）、血磷（PHOS）、胰淀粉酶（AMYL）及肌酐（CRE）明显升高。血气指标中GLU、BUN、钾离子（K⁺）均比参考值高;钠离子（Na⁺）、氯离子（Cl^-）、二氧化碳分压（PCO₂）比参考值降低。犬钩端螺旋体抗原检测板检测结果为阳性。治疗后血常规中白细胞总数（WBC）、淋巴细胞（SCR）比正常参考值明显升高;红细胞总数（RBC）、血红蛋白（HGB）及血小板总数（PLT）则比参考值降低。经Hotelling T²对治疗前后各指标进行多变量分析,结果F=1 905 045.32,P=0.001,差异极显著;单变量检测指标中WBC、W-LCR、SCR、MID、HGB、PCV、MCV、RDW-SD差异极显著（P<0.01）,W-MID、RBC、MCH及血小板分布宽度（PDW）差异显著（P<0.05）。治疗后生化检测指标中,与正常参考值相比,多数指标在正常值范围内。治疗后期病犬精神好转,经Hotelling T²与治疗前差异性分析,多变量F=85.21,P=0.081,差异不显著;单变量检验指标GLU、BUN、PHOS、AMYL及CRE差异极显著（P<0.01）。治疗后期血气指标中GLU、BUN、K⁺均比参考值高;Na⁺、Cl^-及PCO₂比参考值低,对治疗前后血气指标进行Hotelling T²差异性分析,多变量检验结果F=7 978.48,P=0.008,差异极显著;单变量检验结果中,GLU和BUN差异极显著（P<0.01）。治疗方案中,采用利尿、抗生素药物,附加补充电解质、能量、维生素及CoA等综合输液治疗,7 d后,病犬精神好转,机体机能基本恢复正常,转归期部分指标的升高属于机体机能恢复健康的一种正常体现。说明治疗方案正确,综合体液疗法是治疗该病引起肾功能衰竭有效的方法。     

Diagnosis and Therapy on Renal Failure Caused by Canine Leptospira

To determine the pathogen and related symptoms caused by renal failure,and to provide a reasonable diagnosis and treatment plan,a case of disease on Welsh Corgi dog was tested by the method of symptoms,blood routine examination,blood biochemical test,blood gas analysis and identification of pathogen.The results showed that these data of W-SCR,W-MID,MID and RDW-CV rose than normal reference index in blood routine examination before the hospitalization,respectively.Meanwhile,data of W-LCR,PCV,MCV,MCH and RDW-SD had reduced separately.These data of Ca²⁺,GLU,BUN,PHOS,AMYL and CRE were up than normal reference index in blood biochemical test,respectively.These data of GLU,BUN and K⁺ had risen respectively,nevertheless,these data of Na⁺,Cl^- and PCO₂ had lowered separately.The result was positive by the antigen testing with the Leptospira detector plate.After treatment with synthetical therapy,WBC and SCR had risen than normal references index,and data of RBC,HGB and PLT were down respectively.By the Hotelling T² analysis of the difference between before and after the therapy on blood routine index,it was significant difference in the multivariate test (F=1 905 045.32,P=0.001).These data of WBC,W-LCR,SCR,MID,HGB,PCV,MCV and RDW-SD were extremely significant differences in single variable analysis (P<0.01),and other data with significant differences were W-MID,RBC,MCH and PDW(P<0.05).But,in blood biochemical test,most of data were in normal reference values.The mental state of sick dog was recovering gradually normal than before.Multivariate test was no significant difference by the Hotelling T² analysis (F=85.21,P=0.081),but other data of GLU,BUN,PHOS,AMYL and CRE were extremely significantly different in single variable analysis (P<0.01).These data of GLU,BUN and K⁺ were up than after therapy in blood gas analysis,and data of Na⁺,Cl^- and PCO₂ reduced respectively.There were extremely significant differences in the multivariate test (F=7 978.48,P=0.008).These data with extremely significant differences were GLU and BUN in single variable analysis(P<0.01).The sick dog was recovering normal after treatment,and mental state went back to normal after 7 days therapy using the drugs of diuretics,antibiotics,rehydration electrolyte,energy medicine,vitamins and CoA.It should be noted that the part of high index of reexamination was a normal manifestation when the body was recovering to normal functioning.The method of synthetical fluid therapy was provided the correctness to treat the renal failure caused by canine Leptospira.     

Brucellosis in rabbits induced by an experimental infection with Brucella suis germs

Hares were experimentally infected with Brucella suis germs, biotype 2, to study the development of morphological changes in the organs. It was demonstrated that conjunctival and intranasal infection caused morphological brucellosis changes in liver. After conjunctival infection affections were observed in 16 days, and in 27 days after intranasal infection. The morphology of changes did not differ from those occurring in spontaneous brucellosis. The results of morphological examination corresponded to the serological results. It was proved by the results of the experiment that the course of brucellosis could be chronic and that in the process of the natural infection of hares with Brucella suis germs the conjunctival and intranasal modes of infection played an important role. Hares were highly sensitive and responsive to the conditions of the environment where experimental infection was performed. In laboratory conditions the health condition and functions of the organs of hares were impaired by a complex of negative, mainly psychogenic stress factors, which resulted in the death of hares and the experiment had to be finished prematurely on the 41st day after infection.     

氟喹诺酮类药物胶体金试纸的研制

用针对抗氟喹诺酮类(fluoroquinolones,FQs)药物的广谱性单克隆抗体建立了可以同时检测11种FQs药物的胶体金免疫层析方法。该试纸条用20 nm的胶体金标记单抗,将诺氟沙星－卵清蛋白喷涂在检测线上,羊抗小鼠二抗喷涂在质控线上。该胶体金试纸对猪肉和虾中环丙沙星、恩诺沙星和氧氟沙星的检测限都是30 ng/g,对诺氟沙星、培氟沙星、依诺沙星、麻保沙星、洛美沙星、达氟沙星、沙拉沙星和二氟沙星8种药物在以上两种样品中添加浓度为100 ng/g时都可被检出,整个检测过程包括样品前处理的时间可在20 min内完成。试验结果表明符合对这11种FQs药物在鸡肉和虾中残留现场大量筛查的要求。     

Seroprevalence of Canine Herpesvirus-1 and Brucella canis in Finnish Breeding Kennels with and without Reproductive Problems

We compared the serological status of Brucella canis and canine herpesvirus-1 (CHV-1) in Finnish breeding kennels with and without reproductive problems. Dogs from kennels with reproductive problems had significantly higher CHV-1 titres than dogs from kennels having no reproductive problems (p < 0.001). In dogs from kennels with reproductive problems 100% (32/32) had positive titres, whereas in dogs from kennels without reproductive problems 65% (22/34) had positive titres. The median titre for dogs from kennels with reproductive problems was 1 : 160 and for dogs from kennels without reproductive problems 1 : 80. The high prevalence of positive CHV-1 titres in this study indicates that prevention of the disease is difficult and reinforces the need to minimize the reproductive problems caused by CHV-1. All 388 dogs from 94 kennels had negative B. canis titres.     

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.     

奶牛布鲁氏菌病微量凝集试验诊断方法的建立

为探索操作简便、快速、实用的奶牛布病诊断方法,本研究建立了奶牛布鲁氏菌病(布病)诊断96孔V型板微量凝集方法,并优化了试验条件。结果显示,抗原的最佳稀释浓度为1:20,反应最适温度为37℃,反应时间为24 h;为验证本研究建立的微量凝集方法的准确性,对120份奶牛布病虎红平板凝集试验阳性血清样品进行微量法凝集和试管凝集试验,结果显示,微量法与虎红平板凝集试验的符合率为83.3%,试管法与虎红平板凝集试验符合率为80.0%。提示,微量凝集试验可以作为试管凝集试验的替代方法,应用于规模化奶牛场布病的诊断和检疫。     

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.     

Use of an Immunobinding Test on Nitrocellulose Paper to Diagnose Caprine Brucellosis

Veterinary Research Communications -     

口蹄疫病毒通用型金标检测试纸条诊断方法的建立

为了兽医基层工作人员能够快速诊断口蹄疫病原,与其他传染病做鉴别诊断,有必要建立一种快速、简便区分它们的胶体金免疫层析方法。本试验采用柠檬酸三钠还原法制备胶体金。将纯化的Asia 1/China/05流行毒株12S偶联抗原的兔抗体制备免疫胶体金,喷免疫胶体金于玻璃纤维制成金标垫。将纯化A/AF72流行株12S偶联抗原的兔抗体和适量O/China/99流行毒株12S偶联抗原的兔抗体混合固定于硝酸纤维素膜上作为检测带。将羊抗兔抗体固定在硝酸纤维素膜的不同区域作为质控带,并组装成口蹄疫病毒通用型金标检测试纸条。利用其对阴性参考样品、各型阳性参考样品、已知背景的田间样品进行检测。灵敏度试验结果显示:建立的试纸条方法敏感性高、可检测到病毒最低含量为0.98×104 LD50;特异性试验显示:在检测与口蹄疫临床症状相似病原———猪水泡病病毒(SVDV)、水泡性口炎(VS)、水泡性疹(VES)抗原时无交叉反应;重复性试验显示:不同批次间、同一批次内检测结果完全一致;符合性试验显示:检测国家口蹄疫参考实验室已确定口蹄疫病毒血清型的样品90份,阳性符合率、阴性符合率分别为96.70%、100%。本试验研发的口蹄疫病毒通用型金标检测试纸条具有很好的灵敏度、特异性、重复性和符合性,适合国内流行的口蹄疫病原的鉴别诊断。     

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.     

奶牛布氏杆菌病的血清学诊断

为了解虎红平板凝集试验(RBPT)、试管凝集试验(SAT)和酶联免疫吸附试验(ELISA)三种血清学方法在奶牛布氏杆菌病诊断方面的实际应用情况,本试验采集了10份疑似奶牛布氏杆菌病的血清样本,同时采用上述三种血清学诊断方法进行了对比研究。结果显示,RBPT、SAT和ELISA的阳性检出率分别为70%、80%和90%,以ELISA的阳性检出率为最高。因此,具备特异、灵敏、快速等优点的ELISA血清学诊断方法更适合奶牛布氏杆菌病的抗体检测及其研究。