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.     

Comparison of serological tests forBrucella melitensis infection in sheep

Summary A comparative study of the standard tube agglutination test (SAT), Rose Bengal plate agglutination test and counter immuno-electrophoresis (CIEP) was made on 647 sera from naturally aborting ewes, orchitic, in-contact and apparently healthy sheep with no history of vaccination against brucellosis. No individual test could detect all the 13 known positive reactors (the foetuses of which yieldedBrucella melitensis) but by combination of two tests all 13 were positive. The SAT detected more reactors during the early stage of infection while CIEP performed better in later stages of infection. All these tests may be carried out in a field laboratory at very low cost.

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Resumen Se llevó a cabo un estudio donde se comparó la efectividad de la aglutinación en tubo, la prueba Rosa de Bengala y la contrainmunoelectroforesis en 647 sueros de ovejas que habían abortado naturalmente, machos con orquitis, ovejas en contacto y ovejas aparentemente sanas, sin previa historia de vacunación contra brucelosis. Ninguna de las pruebas utilizadas pudo detectar individualmente todos los 13 reactores positivos (de los fetos de los cuales se aislóBrucella melitensis), pero la combinación de dos pruebas detectó todos los reactores positivos. La prueba de la aglutinación en tubo detectó la mayoría de reactores en los estadíos iniciales de la infección, mientras que la contrainmunoelectroforesis se comportó bien en estadíos iniciales de la infección, mientras que la contrainmunoelectroforesis se comportó bien en estadíos tartíos de la infección. Todas estas pruebas pueden realizarse, en laboratorios de campo a un costo bajo.

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Résumé Les auteurs ont réalisé une étude comparative entre l'agglutination en tube standard (SAT), le test d'agglutination sur lame au Rose Bengale (RBPT) et la contre immunoélectrophorèse (CIEP), à partir de 647 sérums prélevés sur des brebis ayant avorté naturellement ou sur des moutons atteints d'orchite ou ayant été en contact mais restés apparement sains et sans passé vaccinal contre la brucellose. Aucun test individuel n'a pu reconna?tre comme positif l'un des 13 animaux reconnus réagissant (dont les foetus hébergeaientBrucella melitensis), mais par combinaison des deux tests, tous les 13 se sont révélés positifs. Le SAT a décelé plus de réagissants au cours des premiers stades de l'infection. Par contre, le CIEP a été plus sensible dans les derniers stades. Tous ces tests peuvent être réalisés dans un laboratoire de terrain à, un co?t trés bas.

     

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.     

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.     

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.     

Brucella melitensis infection following military duty in Iraq

Brucellosis is a common zoonotic disease worldwide; however, few cases are reported in the US. Brucella melitensis infections are primarily acquired via consumption of high-risk foods or travel to endemic areas. We describe a case of B. melitensis infection in a Tennessee soldier following deployment in Iraq. Initial symptoms included knee and back pain. Culture of an aspirate of the left sacroiliac joint yielded B. melitensis. Genetic analysis indicated that this isolate came from the Middle East. Investigation of laboratory workers identified risky exposures and positive serology prompting post-exposure prophylaxis. Military personnel and other travellers should be advised to reduce risk regarding food consumption and animal contact in endemic areas. Additionally, medical providers should remain vigilant for non-endemic zoonoses among recent travellers.     

An ELISA with Brucella lipopolysaccharide antigen for the diagnosis of B. melitensis infection in sheep and for the evaluation of serological responses following subcutaneous or conjunctival B. melitensis strain Rev 1 vaccination.

An indirect enzyme-linked immunosorbent assay (ELISA) with unpurified Brucella melitensis smooth lipopolysaccharide (S-LPS) as antigen was evaluated for the serological diagnosis of B. melitensis infection in sheep in comparison with the Rose Bengal (RB), complement fixation (CF), radial immunodiffusion (RID), microplate agglutination (MA) and rivanol agglutination (RIV) tests. Tests RB and CF detected as positive each of the 77 sera from B. melitensis-infected animals tested, the RID (74), MA (76) and the RIV (72) were less sensitive. However, all tests compared were negative when 77 sera from Brucella-free rams were tested. While subcutaneous Rev 1 vaccination induced high response levels in any of the tests, low level responses were obtained upon conjunctival vaccination, particularly in ELISA and RID tests.     

Comparison of PCR assay and bacteriological culture method for the detection of Brucella melitensis in stomach content samples of aborted sheep fetuses

The aim of this study was to evaluate the polymerase chain reaction (PCR) assay for detection of Brucella melitensis in stomach content samples of aborted sheep fetuses and to compare its performance with bacteriological culture method. It was also aimed to determine the agreement between PCR and Rose Bengal plate test (RBPT). Materials were collected from aborted sheep from 109 different sheep flocks in the region of Van during the lambing seasons of 2004-2005 and 2005-2006. Stomach contents from 135 aborted sheep fetuses were examined by bacteriological culture and PCR, and 135 sera from these aborting ewes were tested by RBPT. Identification and typing of Brucella strains were performed using standard classification test. B. melitensis biovar 3 was isolated from 26 (19.2%) of foetal stomach contents. B. melitensis was detected by PCR in 29 (21.4%) stomach content samples. Twenty five sera (18.5%) from aborting ewes tested positive by RBPT. The detection limit of B. melitensis 16 M strain by PCR was 1.7 x 10(3) cfu (colony forming units) /ml in spiked stomach contents. Diagnostic sensitivity and specificity of the PCR were detected as 100% and 97.2%, respectively. The agreement between PCR and RBPT was found to be 97%. In conclusion, PCR assay would have an advantage over conventional bacteriological culture method, but in particular for its ability to meet the specificity requirements for the detection of B. melitensis in stomach content samples of aborted sheep fetuses.     

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.     

Survey of brucellosis in goats and sheep in the yemen arab republic: Comparison of tests forBrucella melitensis infection in sheep

Summary Sera from 538 Yemeni goats and 690 Yemeni sheep were screened for brucellosis by the Rose Bengal Plate Test (RBPT) and reactors confirmed by the complement fixation test (CFT) and the serum agglutination test (SAT). The prevalence among goats was 0·4% and among sheep 0·6%. The prevalence among 183 imported goats and sheep was 4·4%. The sensitivity and specificity of three seřological tests available for the diagnosis of brucellosis—CFT, RBPT and SAT—were compared using ovine sera obtained throughout an outbreak of abortion due toBrucella melitensis. The RBPT and the SAT were relatively insensitive compared with the CFT (71 and 44% respectively) and the RBPT was as specific as the SAT when suspicious sera were included. The results suggests that the SAT adds little information when used with other tests and the RBPT has limited applications as a screening test for ovine brucellosis.

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Resumen Se examinaron 538 sueros de cabras Yemeni y 690 de ovejas Yemeni, para detectar brucelosis, mediante la prueba Rose de Bengala (RB) confirmando los reactores con la prueba de fijación del complemento (FC) y de seroaglutinación (SA). La prevalencia en cabras fue de 0.4% y en ovejas de 0.6%. La prevalencia dentro de 183 cabras y ovejas importadas fue de 4.4%. La sensibilidad y especificidad de las tres pruebas serológicas utilizadas, se comparó usando suero ovino obtenido en un brote confirmado de brucelosis porBrucella melitensis. Las pruebas de (RB) y (SA) fueron relativamente insensibles comparadas con la de (FC) (71% y 44% respectivamente), y la de (RB) fue tan específica como la de (SA). Los resultados sugieren que, la prueba de (SA) a?ade poca información cuando se utiliza con otras pruebas, y que la de (RB) tiene aplicación limitada para un diagnóstico de población para brucelosis ovina.

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Résumé Des sérums provenant de 538 chèvres et 690 moutons du Yemen on été analysés pour le dépistage de la brucellose par le test des lames au Rose Bengale (TLRB) et les réactions positives ont été confirmées par le test de la fixation du complément (FC) et le test d'agglutination du sérum (TAS). Le taux de fréquence chez les chèvres était de 0,4 p. 100 et chez les moutons de 0,6 p. 100 Le taux de chez 183 ovins et caprins importés étai de 4,4 p. 100 La sensibilité et la spécificité des trois tests sérologiques utilisés dans le diagnostic de la brucellose—TLRB, FC et TAS—ont été comparés sur des sérums ovins obtenus tout au long d'une épidémie d'avortements dus àBrucella melitensis. Le test au Rose Bengale et le test d'agglutination du sérum se sont montrés relativement peu sensibles comparés à la fixation du complément (7 et 44 p. 100 respectivement) et la spécificité du test au Rose Bengale était égale à celle du test de l'agglutination du sérum lorsque des sérums douteux étaient inclus. D'après les résultats, le TAS, utilisé conjointement avec les autres tests apporte peu d'informations supplémentaires et le TLRB a des applications limitées en tant que test de dépistage de la brucellose ovine.

     

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.     

Treatment of Brucella melitensis infection in mice by use of liposome-encapsulated gentamicin

Liposomes with entrapped gentamicin were used to treat mice with infection attributable to Brucella melitensis. Liposomes bearing positive charge and formed by egg yolk lecithin, cholesterol, and stearylamine were effective in the elimination of B melitensis residing in liver and spleen. Negatively charged liposomes, formed by egg yolk lecithin, cholesterol, and dicetyl phosphate were also effective in suppression of the infection in liver, but were less so in suppression of the infection in the spleen. Free gentamicin was less effective than the encapsulated antibiotic. At 20 hours after administration of gentamicin encapsulated in liposomes, the gentamicin concentrations in liver and spleen were similar, regardless of the charge of the liposomes--neutral, positive, or negative. However, positively charged liposomes were more efficient than were other liposome types for the treatment of brucellosis caused by B melitensis.