Comparison of subcutaneous and conjunctival routes of Rev 1 vaccination for the prophylaxis of Brucella ovis infection in rams

The serological response and protection conferred against Brucella ovis by the Rev 1 vaccine was evaluated in both adult (experiment 1) and young rams (experiment 2) vaccinated either subcutaneously or conjunctivally. In experiment 1 the Rev 1 vaccine protected 55.5 per cent and 100 per cent, respectively, of subcutaneously and conjunctivally vaccinated rams against three consecutive challenges that infected 100 per cent of unvaccinated controls. In experiment 2, Rev 1 protected 100 per cent of rams vaccinated subcutaneously and 70 per cent of those vaccinated conjunctivally against a challenge dose able to infect all the unvaccinated controls. The serological response after vaccination was significantly lower in rams vaccinated conjunctivally than in those vaccinated subcutaneously.     

Validation of a competitive ELISA for diagnosis of Brucella melitensis infection in sheep and goats

A competitive enzyme-linked immunosorbent assay (cELISA) was validated for the serodiagnosis of Brucella melitensis infection in small ruminants using 2108 positive and 2154 negative reference sera from sheep and goats. The optimum cut-off values, offering the highest diagnostic sensitivity (DSn) and diagnostic specificity (DSp), determined by receiver operating characteristic analysis, were at 23.6%, 21.8% and 25.0% inhibition of the conjugate control for sheep, goats and both species, respectively. The DSns of the cELISA for sheep, goats and both species at these cut-off values were 89.2% (95% confidence interval 87.1-91.1%), 74.0% (95% CI 71.4-76.5%) and 77.9% (95% CI 76.1-79.7%), whereas DSps were 96.4% (95% CI 95.2-97.4%), 92.9% (95% CI 91.1-94.3%) and 97.2% (95% CI 96.4-97.8%), respectively. Compared to cELISA, indirect ELISA and fluorescence polarisation assay have higher DSns and DSps. However, the results obtained with the cELISA were in good agreement with those of the complement fixation test (CFT) under field conditions using 5735 sheep and goat sera. The cELISA can be used as an alternative to the CFT for diagnosing B. melitensis infection in small ruminants.     

Temporal ELISA response of rams to Brucella ovis following experimental infection or vaccination

Sera from rams vaccinated with antigens extracted chaotropically from Brucella ovis by potassium thiocyanate treatment were used to optimise a whole-cell, enzyme-linked immunosorbent assay (CELISA) and to monitor the temporal serological response of rams which had been challenged with infected semen by the intranasal or intrapreputial route. Three patterns of CELISA response were detected. Thirteen of 15 rams intranasally challenged did not respond serologically (pattern 1 or nil response). Only one of 15 rams in the intranasal group exhibited a rise and fall response with CELISA (pattern 2), while another showed a rise and surge response (pattern 3). The numbers of rams in the intrapreputial group which displayed a pattern 1 or 2 or 3 response were four, nine and two, respectively. No ram with a pattern 2 response excreted B ovis in the semen or showed any other evidence of infection, whereas rams with a pattern 3 response excreted B ovis in the semen and developed palpable lesions. Intrapreputially challenged rams that were CELISA-positive consistently mounted an antibody response against B ovis about two to four weeks earlier than intranasally challenged rams.     

Comparison of milk-ELISA and serum-ELISA for the diagnosis of Brucella melitensis infection in sheep

Milk and blood samples from 704 lactating ewes were examined for the diagnosis of Brucella melitensis infection by milk-ELISA, serum-ELISA, RBPT, SAT and culture of milk. Of these ewes, 209 were from brucellosis free sheep flock, 443 from brucellosis infected sheep flock and 52 were from private sheep flocks of which status for brucellosis was not known. All the 209 ewes belonging to uninfected sheep flock were found negative in all the tests and of the remaining 495 ewes 105 were positive in serum-ELISA, 103 in milk-ELISA, 92 in RBPT, 85 in SAT, and B. melitensis biovar-1 was isolated from the milk of 29 ewes. Of the 105 serum-ELISA positive ewes, 99 were positive and 6 were negative in milk-ELISA, whereas of the 103 milk-ELISA positive ewes, 4 were negative in serum-ELISA. All together, 99 ewes were positive and 386 were negative in both the assays while 10 ewes yielded variable results. The specificity of milk-ELISA in brucellosis free flock was 100% and sensitivity and positive predictive value were 96.11% and 94.28%, respectively, in infected flocks. The Brucella antibody levels in milk and serum samples as determined by milk-ELISA and serum-ELISA were correlated significantly. The milk-ELISA for brucellosis appears to be an attractive alternative of serum-ELISA particularly in the lactating ewes.     

The use of homologous antigen in the serological diagnosis of brucellosis caused by Brucella melitensis

In the European Union the serological diagnosis of brucellosis caused by Brucella melitensis is performed using the heterologous antigen of B. abortus S99. The possible higher sensitivity or ability of an early detection of antibodies by a homologous antigen may prove very useful in the final phases of an eradication programme. Results obtained in sheep experimentally infected by B. melitensis biovar 3 were compared using B. abortus S99, B. melitensis M1, M2 and M3 antigens in the Rose Bengal plate test (RBPT), the complement fixation test (CFT) and an enzyme-linked immunosorbent assay (ELISA) test. Forty-six sheep from an officially brucellosis-free flock were experimentally infected intraconjunctivally with B. melitensis biovar 3. Prior to infection, all animals were tested first against Brucella antibodies, weekly for 2 months post-infection (PI) and then monthly for a further 7 months. All sera were tested against the antigens listed above using RBPT, CFT and ELISA. Using a Bayesian approach, test sensitivities were estimated and compared. Their ability for the early detection of antibodies was evaluated through a regression model based on a logit response model, using the number of days PI as the independent variable and the logit of the fraction of positive animals as the dependent variable. No significant differences were detected among the various antigens used, either in terms of sensitivity or in terms of antibody kinetics; however, the CFT was significantly less sensitive than the RBPT and ELISA and it also showed a lower rate of increase of percentage positive animals (beta-coefficient of regression analysis).     

Control of brucellosis in Kuwait by vaccination of cattle,sheep and goats withBrucella abortus strain 19 orBrucella melitensis strain Rev. 1

Summary In Kuwait, approximately 12,000 diary cows were vaccinated with a reduced dose of 3×10⁹ Brucella abortus strain 19 and approximately 350,000 sexually mature sheep and goats with a reduced dose of 10⁷B.melitensis strain Rev. 1. Using the criteria of prevaccinal and postvaccinal incidences of antibodies, abortions, and human cases of brucellosis, the programme was very successful. Widespread vaccination of adult animals is the most effective method of controlling brucellosis among cattle, sheep and goats in many countries.

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Resumen En Kuwait, se vacunaron aproximadament 12,000 vacas lecheras con una dosis reducida de 3×10⁹ organismos deBrucella abortus cepa 19 y approximadament 350,000 ovejas y cabras sexualmente maduras fueron vacunadas con una dosis reducida de 10⁷ organismos deB. melitensis opa Rev. 1. Utilizando los criterios prevacunales de incidencia de anticuerpos, abortos, y casos humanos de brucelosis, el programa fuvo gran exito. Ef método mas efectivo de control de la brucelosis bovina, ovina y caprina an numerosas paises es la amplia utilización de vacuna en animales adultos.

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Résumé Au Kuwait, environ 12,000 vaches laitiěres ont été vaccinées avec une dose réduite de souche 19Brucella abortus (3×10⁹) et environ 350,000 moutons et chevres qui étaient matures sexuellement, one été vaccinés avec une dose réduite de soucheB. melitensis Rev. 1 (10⁷). Basé sur la présence d'anticorps, sur le nombre d'avortements et de cas de brucellose chez les humains avant et aprés vaccination, le programme a été un succěs. La méthode la plus effective pour contr?ler la brucellose chez les bovins, les ovins et les caprins dans beaucoup de pays et de vacciner sans exception tous les animaux adultes.

     

Assessment of serological response of young and adult sheep to conjunctival vaccination with Rev-1 vaccine by fluorescence polarization assay (FPA) and other serological tests for B. melitensis

The serological response of young and adult sheep vaccinated conjunctivally with Rev-1 vaccine was assessed by fluorescence polarization assay (FPA), Rose Bengal test (RBT), complement fixation test (CFT), modified Rose Bengal test (m-RBT), indirect ELISA (i-ELISA) and competitive ELISA (c-ELISA), at different post vaccination intervals. One hundred and thirty six adult sheep and 64 lambs were used in the study. The vaccinated animals were bled prior to vaccination (0 day) and thereafter at 21st, 42nd, 35th, 63rd, 91st, 125th, 159th, and 223rd and 330th day post vaccination. The majority of animals (young and adult) showed positive reaction by FPA, RBT, CFT, m-RBT and c-ELISA 21 days post vaccination, whereas by i-ELISA at 42 days. All tests perform equal when animals vaccinated as young are tested 125 days (4 months) post vaccination. In case of animals vaccinated at adulthood, FPA, RBT, CFT and c-ELISA perform equal if the animals are tested 223 days (approximately 8 months) post vaccination. I-ELISA and m-RBT show low specificity if ewes vaccinated at adulthood are tested 330 days (11 months) post vaccination. If control of brucellosis in sheep is based on conjunctivally vaccination of lambs with Rev-1, the vaccinated animals can be tested by any test used for diagnosis of B.melitensis infection accurately at least 4 months post vaccination. If brucellosis control is based on mass vaccination the use of m-RBT and i-ELISA is not recommended for testing adult animals at least for 330 days (11 months) post vaccination due to tests low specificity. Further research is needed so the appropriate cut-offs to be established for FPA, c-ELISA or i-ELISA to become valuable tools for the eradication of Brucella spp. infection in small ruminants in areas where vaccination is practiced.     

Seroprevalences and lack of abortions after vaccination of Churra sheep with reduced doses of Rev. 1 Brucella melitensis vaccine by subcutaneous or conjunctival routes

A field study to evaluate the serological response and the safety of different doses and administration routes of the Rev. 1 vaccine was carried out on two Churra breed flocks. Reduced doses of 2.3 × 10⁶ and 3 × 10⁷ live organisms were administered by the subcutaneous or the conjunctival route, respectively. In those animals which were seropositive before vaccination, the percentage of positive sera declined progressively in a similar way in all groups over the 36 weeks that the study lasted; the antibody titers also dropped continuously in the group vaccinated by the conjunctival route with the lower dose, while in the remaining three groups there was a transitory increase in the 4th week after vaccination. In those animals which were serologically negative prior to vaccination, the percentage of positive sera and the antibody titers generally reached their peak in the 4th week after vaccination, followed by a progressive decline in succeeding weeks. Similarly, titers were higher in animals vaccinated subcutaneously than in those vaccinated by the conjunctival route. The differences between the frequencies of positive sera and the levels of antibodies were important when routes were compared. Animals receiving a dose of 2.3 × 10⁶ CFU subcutaneously had a satisfactory serological response, with a more rapid decline in their level of antibodies than in the animals which were vaccinated with 3 × 10⁷ CFU by the same route. No cases of abortion were reported in the 461 vaccinated ewes.     

The isolation and serology of the "FSA" Brucella melitensis Rev. 1 mutant in a flock of sheep

A flock of sheep, known to be infected with the "FSA" mutant of Brucella melitensis Rev. 1, was examined serologically and bacteriologically to determine whether any relationship existed which would help in the control of this infection in the field. An attempt was also made to determine whether vertical transmission occurred. Twenty-one out of 62 sheep were bacteriologically positive. The best organs for isolation were the udder, supramammary lymphnodes and uterus. No significant relationship could be shown between the complement fixation test and bacterial isolation. The absence of any relationship between serological and bacteriological results agrees with a short-lived infection. None of the 24 lambs sacrificed at 5 months showed either serological reactions or were bacteriologically positive, thus no vertical transmission could be shown.     

羊种布鲁氏菌疫苗株PCR-RFLP鉴定方法的建立

为建立一种羊种布鲁氏菌Rev.1疫苗株PCR-RFLP鉴定方法,利用羊种布鲁氏菌Rev.1具有链霉素抗性的特性,选取与链霉素抗性相关编码核糖体蛋白S12的rps L基因为模板设计引物,经PCR扩增后,将产物进行Nci I酶切,发现羊种布鲁氏菌疫苗株Rev.1出现约500 bp条带,而不具有链霉素抗性的羊种布鲁氏菌株16M则出现384 bp条带。结果表明,PCR-RFLP方法能够用于羊种布鲁氏菌Rev.1疫苗株的鉴定,这为今后区分羊种布鲁氏菌疫苗株Rev.1免疫与野株感染提供了基础。     

Evaluation of a modified Rose Bengal test and an indirect enzyme-linked immunosorbent assay for the diagnosis of Brucella melitensis infection in sheep

A modified Rose Bengal test (mRB) and an indirect ELISA (iELISA) with Protein G as the conjugate, were evaluated for the diagnosis of Brucella melitensis infection in unvaccinated sheep with a known bacteriological status, and their diagnostic efficacy was compared with that of the standard Rose Bengal (RB) and Complement Fixation (CF) tests used in the current eradication campaign in EU countries. All tests showed 100% specificity when testing the sera from 212 Brucella-free sheep. When testing the sera from 219 Brucella melitensis culture-positive sheep, both the mRB and iELISA tests were more sensitive (98.6% and 96.8%, respectively) than the RB and CF tests (95.0% and 92.7%, respectively). These results were similar when testing the sera from 181 animals belonging to infected flocks but found bacteriologically negative, suggesting that the mRB or iELISA tests could advantageously replace the current RB procedure used as the screening test.     

Evaluation of different enzyme-linked immunosorbent assays for the diagnosis of brucellosis due to Brucella melitensis in sheep

Six serological assays for the diagnosis of ovine brucellosis, due to Brucella melitensis were evaluated. Reference serum samples from sheep of known B. melitensis infection status (n = 118) were assessed using the Rose Bengal test (RBT), complement fixation test (CFT) and four commercial enzyme-linked immunosorbent assays (ELISAs), including two indirect ELISAs (iELISAs), one competitive ELISA (cELISA) and one blocking ELISA (bELISA).The highest differential positive rates (DPR) were obtained with the cELISA and bELISA, while the lowest DPR was estimated using iELISAs. A latent class analysis was performed to estimate the accuracy of the CFT, RBT and bELISA using 1827 sera from sheep undergoing testing as part of a surveillance and control programme. Lower sensitivity and specificity were obtained for the three serological tests when the field samples were used. A higher DPR was achieved by the CFT, compared to bELISA and RBT. The results suggest that ELISAs, and particularly the bELISA, might be suitable for inclusion in the European Union legislation on intra-community trade for diagnosing B. melitensis infection in sheep, as it has a similar test performance compared to the RBT.     

An evaluation of indirect immunofluorescence in the serological diagnosis of Nosema cuniculi infection.

An indirect immunofluorescence test for serum antibodies to Nosema cuniculi was evaluated in rabbits by comparison with established histopathological methods of diagnosis. Cell-culture isolation procedures and immunofluorescence and histological techniques designed to detect the parasite in urine or tissues were used to confirm the diagnosis. The serological diagnosis showed excellent correlation with presence of brain lesions characteristic of nosematosis, and N cuniculi were detected in 63 per cent of seropositive rabbits but in no seronegatives. Serum cross-reactivity tests between N cuniculi and Toxoplasma gondii showed that these two protozoa are antigenically distinct. The technique offers a simple, sensitive and reliable procedure for diagnosis of nosematosis in a living animal.     

Single-step purification and evaluation of recombinant BP26 protein for serological diagnosis of Brucella ovis infection in rams

To investigate the value of the BP26 protein in the serological diagnosis of ovine brucellosis caused by Brucella ovis, recombinant BP26 protein was produced in Echerichia coli and purified for use in an indirect enzyme-linked immunosorbent assay (I-ELISA). The majority of the recombinant protein was recovered from the supernatant of sonicated recombinant E. coli cells in a soluble form. This facilitated the purification of the recombinant BP26 protein which was achieved by using ion-exchange chromatography. After one step of purification, the purity of the recombinant BP26 protein was analyzed by using SDS-PAGE, Coomassie blue staining, and Western blot with a monoclonal antibody (MAb) directed against the BP26 protein. The degree of purity appeared satisfactory so that it could be directly used in I-ELISA. Although the recombinant BP26-ELISA appeared less useful than I-ELISA using the B. ovis hot saline (HS) extract as antigen, the high number of sera from B. ovis infected rams found positive (90%) in the recombinant BP26-I-ELISA indicated that the BP26 protein may be an additional suitable antigen for serological diagnosis of B. ovis infection in rams.