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.     

Comparison of three serological tests for Brucella ovis infection of rams using different antigenic extracts

The sensitivity and specificity of the complement fixation, gel diffusion and ELISA tests for the diagnosis of Brucella ovis infection of rams have been compared using three different antigenic preparations. The antigens obtained by petroleum ether - chloroform - phenol, or cold saline extractions gave poorer diagnostic results than those obtained by hot saline extraction in all the tests. The best sensitivity was obtained with the ELISA (97.6 per cent) followed by the gel diffusion (96.4 per cent) and complement fixation tests (92.7 per cent). The gel diffusion test detected as positive the two rams negative in the ELISA, while the complement fixation test did not improve the sensitivity of the other tests. Under these conditions all the tests were 100 per cent specific when testing sera from rams free of B ovis.     

An indirect haemagglutination test for the detection of Brucella ovis antibodies

Abstract

Extract

Surveys on perinatal infection in lambs in New Zealand have been reported and the pathology and bacteriology of the conditions described (Hartley and Boyes, 1955 Hartley, W. J. and Boyes, Betty W. 1955. Proc. N.Z. Soc. anim. Prod., 15: 120–120.  [Google Scholar], 1964 Hartley, W. J. and Boyes, Betty W. 1964. N.Z. vet J., 12: 33–33. [Taylor &; Francis Online] , [Google Scholar]; McFarlane, 1955 McFarlane, D. 1955. Proc. N.Z. Soc. anim. Prod., 15: 104–104.  [Google Scholar]; Hartley and Kater, 1964 Hartley, W. J. and Kater, Joan C. 1964. N.Z. vet. J., 12: 49–49. [Taylor &; Francis Online] , [Google Scholar]). Potentially pathogenic organisms were isolated from 58 to 288 lambs from five flocks, Clostridium septicum being isolated from five of these cases (Hartley and Boyes, 1955 Hartley, W. J. and Boyes, Betty W. 1955. Proc. N.Z. Soc. anim. Prod., 15: 120–120.  [Google Scholar]). In another survey, 5.5% of lambs born dead or dying up to 4 weeks of age died from navel infection. Clostridium septicum was isolated from 69% of 48 consecutive cases (Hartley and Boyes, 1964 Hartley, W. J. and Boyes, Betty W. 1964. N.Z. vet J., 12: 33–33. [Taylor &; Francis Online] , [Google Scholar]). McFarlane (1955 McFarlane, D. 1955. Proc. N.Z. Soc. anim. Prod., 15: 104–104.  [Google Scholar]) recorded that 7.3% of perinatal mortality was due to navel infection but no bacteriology was carried out nor was the organism suspected stated. On individual farms, up to 15% of lambs recorded died from navel ill. It should be pointed out that, in this survey, only small numbers of lambs were received from some properties.     

Identification of immunoreactive membrane antigens of Brucella ovis by ELISA profiling

Enzyme-linked immunosorbent assay (ELISA) profiling was used to identify the immunoreactive membrane antigens of Brucella ovis. Immunoreactive membrane antigens obtained after detergent extraction of the bacterial membrane complex (inner and outer membranes) were resolved into five peaks (A, B, B1, C and D) by gel permeation chromatography. Aliquots from each of the chromatographed fractions were coupled to 96-well microtitre plates and immunoreactive fractions identified with sera from two rams. Serum from ram 1 which had been vaccinated with a single injection of formalin-killed B ovis emulsified in incomplete Freund's adjuvant identified A and B as the major immunoreactive peaks. Serum from ram 2, which had been successfully infected with B ovis, reacted mainly against peaks A, B1, C and D. This observation facilitated the use of A, B, B1, C and D peak antigens as test reagents to examine the serological response of 12 other rams exposed to B ovis by vaccination or intraconjunctival or intravenous inoculation. Sera from rams which developed productive infections reacted strongly against peaks A, B1, C and D while vaccinated rams had preferential antibody activity against peaks A and B.     

Detection of antibodies against Brucella in the aqueous humor using ELISA

In aqueous bulbi of experimentally against Brucella abortus immunized guinea pigs and rabbits specific antibodies could be detected by ELISA-tests. This first demonstration of brucella antibodies in the aqueous shows that aqueous can be used for serological examinations. For practical use more investigations in experimental animals are necessary, e.g. on kinetics of orbital antibodies absence of interfering factors and biomechanics of proteins in aqueous.     

Comparison of an indirect ELISA with the Brucella milk ring test for detection of antibodies to Brucella abortus in bulk milk samples.

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of Brucella abortus antibodies in bovine bulk milk samples was evaluated. About 31 individual milk samples from B. abortus infected cows were diluted into bulk milk from a brucellosis free herd. Individual milk samples obtained from 96 negative or positive herds to ELISA or Brucella ring test (BRT), were tested by ELISA. All positive cows were bled and serum samples were tested by the complement-fixation test (CFT) which was considered the definitive test. A herd was considered infected if at least, one cow was positive in the CFT. Four samples were negative in the BRT at the dilution 1:10 but positive in the ELISA. For samples positive in both tests, BRT titers ranged from 1:10 to 1:480 while ELISA titers ranged from 1:10 to 1:3200.Using bulk milk samples, the sensitivity of the ELISA (98.1%) was higher than the BRT (72.2%) but the specificity of BRT (90.5%) was not statistically different (P=1.0) from the ELISA (88.1%). The implications of the results for brucellosis control are discussed.     

Comparison of ELISA and CFT assays for Chlamydophila abortus antibodies in ovine sera

OBJECTIVE: To compare the sensitivity and specificity of Chlamydophila abortus antibody assays, to find a suitable serological assay for testing sheep for export. DESIGN: Comparison of results from known positive and negative sheep populations. PROCEDURE: Fifty-five positive and fifty negative sera were analysed by four enzyme linked immunosorbent assays (ELISA), three using recombinant antigens based on the chlamydial polymorphic outer membrane proteins (POMP90-3, POMP90-4, POMP80-90) and one using a synthetic peptide based on chlamydial major outer membrane proteins (MOMP-P). They were also analysed by complement fixation tests (CFT) using crude antigens from chlamydia isolated from an Australian sheep, a Californian parakeet and a Texan turkey. Assay sensitivity and specificity were expressed as point estimates and 95% confidence intervals. Results were compared using McNemar's test for paired samples. RESULTS: ELISA sensitivity ranged from 70 to 98% and complement fixation test sensitivity from 60 to 96%; with POMP90-3 > POMP90-4 > CFT (parakeet) > CFT (turkey) > POMP80-90 > MOMP-P > CFT (sheep). There was no significant difference from POMP90-3 to POMP80-90 (P > 0.05). ELISA specificity ranged from 88 to 100% and CFT specificity was 100% for all three antigens; with CFT and POMP90-4 > MOMP-P > POMP80-90 > POMP90-3. There was no significant difference from CFT to POMP80-90 (P > 0.05). Changing the CFT cut-off from 1:32 to 1:4 substantially reduced the specificity with little improvement in sensitivity. CONCLUSION: Assays using POMP90-4, POMP80-90, CFT (parakeet) and CFT (turkey) had equivalent sensitivity and specificity; none of the ELISAs were more specific than any CFT. The POMP80-90 ELISA is recommended as an alternative to CFT (parakeet) but as its specificity is not ideal the search for a more specific assay should continue.     

The persistence of antibodies against Brucella ovis and Brucella abortus in rams following vaccination: A field study

Primary hypoparathyroidism was diagnosed in three St. Bernard bitches. Anorexia, behavioural changes, muscle tremors, seizures, panting respiration, and cataracts were the clinical signs observed. The serum concentration of calcium was low, the phosphorus concentration elevated, and the immunoreactive parathyroid hormone level low in all dogs.

The aetiology of the hypoparathyroidism was not determined in any of the dogs. Treatment with synthetic vitamin D (1,25-dihydroxycholecalciferol) and an oral calcium supplement was successful in restoring and then maintaining a normal concentration of serum calcium in two of the dogs.     

Comparison of the enzyme-linked immunosorbent assay and complement fixation test for detecting Brucella ovis antibodies in sheep

A solid-phase, indirect, enzyme-linked immunosorbent assay (ELISA) was compared with the microtitre complement fixation test for detecting Brucella ovis antibodies in 220 ram sera. The ELISA was more sensitive than the complement fixation test; it demonstrated antibodies in 11 sera from known infected or vaccinated rams that were complement fixation test negative. No false positives were recorded with the ELISA and, in 36 sera positive to both tests, the ELISA titres were consistently higher than the corresponding complement fixation test titres.     

丝状霉形体山羊亚种血清抗体间接ELISA检测方法的建立

利用丝状霉形体山羊亚种标准株PG3制备抗原,经纯化后作为包被抗原建立了间接ELISA方法,用于检测丝状霉形体山羊亚种血清抗体。经方阵滴定确定最佳抗原包被浓度为1.09μg/mL,血清样品最佳稀释度为1∶64,兔抗羊IgG辣根过氧化物酶结合物的最佳稀释度为1∶40 000,抗原抗体最佳结合时间为1 h。判定标准为样品D490 nm值与标准阴性血清D490 nm值之比（P/N）≥3为阳性,≤2.5为阴性,介于二者之间的为可疑。重复性和稳定性试验证明,建立的间接ELISA的稳定性和重复性良好,与间接血凝试验相比,其敏感性较高。     

Use of enzyme-linked immunosorbent assay for detection of antibodies to Brucella ovis in sheep: field trial

An enzyme-linked immunosorbent assay (ELISA), using an autoclave-extracted soluble antigen, for the detection of serum antibodies to Brucella ovis in sheep was developed. The test seemed to be both sensitive and specific, on the basis of the control groups studied. The antigen showed no deterioration in prepared plates stored at -70 C for up to a year. The ELISA was used in conjunction with palpation of rams for epididymal lesions as a means to detect and control B ovis infection in a naturally infected flock. All rams were evaluated by the ELISA. At the time that the first blood sample was obtained, all positive and suspicious reactors were removed. With subsequent blood sample collections, only the positive reactors were removed. Brucella ovis was not isolated from any rams during the following year, and none of the mature breeding rams developed epididymal lesions.     

马冠状病毒抗体的间接ELISA检测方法的建立

将含有马冠状病毒N蛋白(ECV-N)的重组杆状病毒rBac-ECV-N感染Sf9昆虫细胞,以表达的ECV-N蛋白作为抗原,以酶标记的鼠抗马IgC特异性单抗为二抗,建立了检测马冠状病毒抗体的ELISA方法.研究结果表明,ECV-N蛋白最佳包被浓度为10μg/mL初提重组蛋白,马血清以1:100稀释,酶标记的抗马IgG单克隆抗体工作浓度1:5 000.可以检出抗马冠状病毒血清抗体.利用该方法对来自3个国家的1064份马血清进行了检测,检测结果表明:国内711份马血清中检测出13份阳性;澳大利亚335份马血清中检测出31份阳性;瑞典18份马血清均为阴性.该间接ELISA方法的建立,为马冠状病毒感染的预防和控制,出入境检验检疫提供了一种新的检测方法.     

The detection of Brucella ovis infection in rams

Abstract

Extract

Each year in New Zealand, many rams are palpated for lesions of the epididymes and testes. Although regarded initially as a method of detecting infertile rams (Crawford and James, 1950 Biberstein, E. L. and McGowan, B. 1958. Cornell Vet., 48: 31–31.  [Google Scholar]), the technique is now regarded primarily as a method of detecting infection with the organism for which Buddie (1956 Buddle, M. B. 1956. J.Hyg., 54: 351–351.  [Google Scholar]) proposed the name Brucella ovis.     

Immunization with Brucella melitensis Rev 1 against Brucella ovis infection of rams

The efficacy of Brucella Melitensis Rev 1 vaccine (Rev 1) for the prophylaxis of Brucella ovis ram epididymitis was evaluated. Twenty-nine 3-month-old rams were vaccinated with 2 X 10(9) Rev 1 and 14 were revaccinated with 5 X 10(8) at 14 months of age. Six rams remained unvaccinated as a control group. All rams were challenged with 5 X 10(8) B. ovis at 21 months of age. Before being slaughtered 8 weeks later, only one vaccinated ram developed epididymitis while four of the six control rams developed testicular alterations. Genital and selected extragenital organs and lymph nodes were removed at slaughter and inoculated on selective media. B. ovis was isolated from 26.6% of the vaccinated rams, 21.4% of the revaccinated rams and 100% of control rams. Portions of epididymis, testes and vesicular glands were also used for pathological studies. More severe lesions were observed in control rams than in vaccinated ones. In conclusion, these results show that vaccination of young lambs, followed or not by revaccination, is a suitable method for the prophylaxis of B. ovis infection of rams.     

猫杯状病毒抗体间接ELISA检测方法的建立

将构建的猫杯状病毒(Feline Calicivirus,FCV)衣壳蛋白VP1重组表达质粒转化感受态细胞,以表达的FCV-VP1蛋白作为包被抗原,以酶标记的兔抗猫IgG作为二抗,建立检测猫杯状病毒抗体的间接ELISA检测方法。确定了最佳封闭液、最佳血清稀释液、最佳酶标二抗稀释液,当酶标记的兔抗猫IgG效价为1∶20 000时可以检出猫杯状病毒血清抗体。利用本实验室建立的检测方法对收集的96份临床血清进行检测,结果表明96份猫血清中检测出40份阳性。     

马疱疹病毒抗体间接ELISA检测方法的建立

为建立一种检测马疱疹病毒抗体间接ELISA方法,采用纯化后的马疱疹病毒1型和马疱疹病毒4型g D蛋白作为抗原,经方阵滴定法优化ELISA反应条件,并对该方法进行特异性和重复性试验。结果显示,使用此方法检测其他马常见病毒病阳性血清均为阴性;组内、组间变异系数均小于10%。采用建立的方法与国外商品化的马疱疹病毒诊断试剂盒对送检的300份马血清进行检测比较,两者符合率为95%。结果表明,本次试验建立的间接ELISA方法可以用来检测马疱疹病毒抗体,为马鼻肺炎的防控工作提供技术支撑。