Comparison of different antigenic preparations for the detection of ovine serum antibodies against Brucella ovis by ELISA

An enzyme-linked immunosorbent assay has been developed for the detection of antibodies against Brucella ovis using serum from control rams (Con-S), naturally infected rams (Inf-S), rams inoculated intravenously with B. ovis (IV-S) and rams vaccinated intramuscularly (IM-S). The serum was titrated by serial double dilutions from 1/25 to 1/25,600 against whole bacteria, B. ovis lipopolysaccharide and a detergent-extracted component of the outer membrane complex of B. ovis as antigens immobilised on microtitre plates. Sheep antibodies bound to antigen were assayed with rabbit anti-sheep gammaglobulin and alkaline phosphatase conjugated protein A. A high level of antibody activity against intact B. ovis cells was detected in Inf-S and IM-S. When lipopolysaccharide was the immobilised antigen, only IM-S yielded significant antibody activity. The component from detergent extracts of the outer membrane complex of B. ovis reacted best with serum (up to 1/6,400) from field-infected rams, while serum from vaccinated and intravenously inoculated rams registered significant titres up to a serum dilution of 1/800 and 1/200 respectively. These results indicate that ELISA is a very sensitive test but its value as a serodiagnostic procedure is dependent upon the choice of antigen used in the assay.     

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.     

Detection of antibodies to Brucella ovis in sheep milk using B. ovis and B. canis antigen

The diagnostic techniques most widely used for detecting brucellosis caused by Brucella ovis are serological tests such as complement fixation (CFT), agar gel immunodiffusion (AGID), and ELISAs. However, to our knowledge, milk tests, with the advantage that samples may be taken in a non invasive manner, have not been investigated as diagnostic tools. We studied 144 samples of milk and sera from lactating ewes, comparing bacteriological studies, serological and milk tests using Brucella canis and B. ovis antigens. A group of 75 ewes in an uninfected flock were serologically negative to rapid slide agglutination test (RSAT), indirect ELISA (IELISA)-B. canis, AGID and IELISA-B. ovis. The milk of these ewes had an IELISA-B. canis mean (%P) value of 16.18 (S.D. 5.63), while the IELISA-B. ovis had a mean (%P) value of 12.52 (S.D. 4.94). A cut-off value of (%P) 27.44 (+2 S.D.) or (%P) 33 (+3 S.D.) was determined by milk-ELISA-B. canis and (%P) 22.4 (+2 S.D.) and (%P) 27.34 (+3 S.D.) by milk-IELISA-B. ovis. These cut-off values were adjusted by receiver-operator characteristics (ROC) analysis using 23 positive samples from infected ewes, which indicated a milk-IELISA-B. canis cut-off value of (%P) 33 and milk-IELISA-B. ovis of (%P) 26 with 100% sensitivity and specificity. Based on our results, we propose that, following a study of a larger number of samples, the milk-IELISA-B. canis could be considered a suitable test for the diagnosis of B. ovis brucellosis in lactating ewes.     

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.     

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.     

Discrimination between sheep antibodies to Brucella melitensis and to Brucella ovis

When preparations containing smooth Brucella abortus lipopolysaccharide (LPS) were used as antigens in an ELISA, strong positive reactions were obtained with sera from sheep infected with Brucella melitensis or with Brucella ovis. Oxidation of the LPS with sodium metaperiodate greatly reduced the extent of the cross-reactions with antisera to B. ovis, with little effect on the reactions with antisera to smooth B. melitensis. Periodate oxidation of hot saline extract (HSX) antigen of B. ovis markedly reduced its reactivity in ELISA with anti-B. ovis sera and eliminated cross-reactivity with anti-B. melitensis sera. The reactivity of HSX was maintained after treatment with proteinase K.

A simple ELISA system, in which replicate samples from a single serum dilution were tested in parallel against both B. ovis HSX antigen and periodate-oxidised smooth phase B. abortus LPS, was evaluated. It was found to discriminate well between antibodies induced by vaccination or virulent infection with B. melitensis strains and those induced by infection with B. ovis.     

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.     

Vaccination against Brucella ovis infection in sheep

Extract

Dairy cows in New Zealand subsist chiefly on pasture or pasture products such as hay and silage, whilst concentrates are fed only in exceptional cases. Town supply herds are concerned with the production of winter milk and payment by the dairy factory is based on their milk production assessment during the months of June, July, and August. It is therefore necessary to have maximum milk production during these winter months when pastures are at their minimum rate of growth. As a food supplement, therefore, many town supply herds augment their feeding with crops and wet brewers' grains. The grains are fed from hoppers in the bail and are also spread out in the paddocks or placed in large wooden troughs. Most grains are purchased on a contract basis from the brewers, more being available during the summer months, when more beer is consumed. Friday's load of grains is usually stored and the consumption spread out over the week-end until the first fresh load is brought in on Monday morning.     

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.     

Immunochemical studies on a Brucella ovis specific protein antigen

A Brucella ovis surface protein antigen (P-II), obtained by gel filtration with Sepharose 4B of a hot saline extract was characterized. The analysis of P-II over gradient sodium dodecylsulfate electrophoresis yielded an 18.5 and a 20 kDa band. In a radioimmunoprecipitation assay using P-II labeled with 125I, the antigen reacted specifically only with sera from rams experimentally infected with a naturally occurring rough strain of B. ovis and did not react with sera from rams experimentally infected with other smooth Brucella strains (B. abortus and B. melitensis).     

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.     

Production of immunity in rams against Brucella ovis infection

Extract

During earlier investigations in New Zealand into the bacterial and mycotic flora of hedgehogs (Erinaceus europaeus), strains of Salmonella typhimurium were obtained from the gut of a number of animals. It was therefore decided to examine these animals more thoroughly for the presence of salmonellae. This paper embodies the results of a small survey subsequently carried out in the Hamilton suburban area during February, 1964.     

狂犬病抗体水平检测方法综述

检测人和动物体内抗狂犬病毒抗体水平主要有以下五个方面作用：①确定人和动物暴露前的抗体水平、预防免疫效果和动物的免疫水平及免疫覆盖率。②检测暴露后进行免疫治疗的人体内的中和抗体水平,以确定暴露后治疗效果。③通过测定狂犬病疑似患者或患畜早期和晚期双份血清中和抗体水平的差异（如抗体水平增高4倍以上）,作为狂犬病确诊的依据之一。④通过测定野生动物狂犬病中和抗体的水平,了解狂犬病在动物种群中隐性感染的情况。     

Lameness in rams following vaccination against Brucella ovis infection

Extract

Since the introduction in 1957 of a vaccination procedure to control Brucella ovis infection in sheep, many thousands of rams have been vaccinated annually throughout New Zealand almost without complications.     

Evaluation in mice of Brucella ovis attenuated mutants for use as live vaccines against B. ovis infection

Brucella ovis causes ram contagious epididymitis, a disease for which a specific vaccine is lacking. Attenuated Brucella melitensis Rev 1, used as vaccine against ovine and caprine brucellosis caused by B. melitensis, is also considered the best vaccine available for the prophylaxis of B. ovis infection, but its use for this purpose has serious drawbacks. In this work, two previously characterized B. ovis attenuated mutants (Δomp25d and Δomp22) were evaluated in mice, in comparison with B. melitensis Rev 1, as vaccines against B. ovis. Similarities, but also significant differences, were found regarding the immune response induced by the three vaccines. Mice vaccinated with the B. ovis mutants developed anti-B. ovis antibodies in serum of the IgG₁, IgG_2a and IgG_2b subclasses and their levels were higher than those observed in Rev 1-vaccinated mice. After an antigen stimulus with B. ovis cells, splenocytes obtained from all vaccinated mice secreted similar levels of TNF-α and IL12(p40) and remarkably high amounts of IFN-γ, a crucial cytokine in protective immunity against other Brucella species. By contrast, IL-1α -an enhancer of T cell responses to antigen- was present at higher levels in mice vaccinated with the B. ovis mutants, while IL-10, an anti-inflammatory cytokine, was significantly more abundant in Rev 1-vaccinated mice. Additionally, the B. ovis mutants showed appropriate persistence, limited splenomegaly and protective efficacy against B. ovis similar to that observed with B. melitensis Rev 1. These characteristics encourage their evaluation in the natural host as homologous vaccines for the specific prophylaxis of B. ovis infection.