Brucella abortus Strain RB51 Vaccine: Immune Response after Calfhood Vaccination and Field Investigation in Italian Cattle Population

Immune response to Brucella abortus strain RB51 vaccine was measured in cattle vaccinated at calfhood. After an increase at day 6 post-vaccination (pv), the antibody level recorded in the 10 vaccinated animals remained constant for two months, and then progressively decreased. All vaccinated animals remained negative from day 162 pv to the end of the study (day 300 pv). Only at days 13 and 14 pv the RB51-CFT showed 100% sensitivity (credibility interval (CI) 76.2%–100%). The results indicate that the possibility to use RB51-CFT for the identification of cattle vaccinated at calfhood with RB51 is limited in time. A field investigation was carried out on 26,975 sera collected on regional basis from the Italian cattle population. The study outcomes indicate that in case of RB51-CFT positive results observed in officially Brucellosis-free (OBF) areas and, in any case, when an illegal use of RB51 vaccine is suspected, the use of the RB51-CFT alone is not sufficient to identify all the vaccinated animals. The design of a more sophisticated diagnostic protocol including an epidemiological investigation, the use of RB51-CFT, and the use of the skin test with RB51 as antigen is deemed more appropriate for the identification of RB51 vaccinated animals

Borexino : geo-neutrino measurement at Gran Sasso, Italy

Geo-neutrinos, electron anti-neutrinos produced in beta-decays of naturally occurring radioactive isotopes in the Earth, are a unique direct probe of our planet's interior. After a brief introduction of the geo-neutrinos' properties and of the main aims of their study, we discuss the features of a detector which has recently provided breakthrough achievements in the field, Borexino, a massive, calorimetric liquid scintillator detector installed at the underground Gran Sasso Laboratory. With its unprecedented radiopurity levels achieved in the core of the detection medium, it is the only experiment in operation able to study in real time solar neutrino interactions in the challenging sub-MeV energy region. Its superior technical properties allowed Borexino also to provide a clean detection of terrestrial neutrinos. Therefore, the description of the characteristics of the detected geo-neutrino signal and of the corresponding geological implications are the main core of the discussion contained in this work

Standardisation of an indirect enzyme-linked immunosorbent assay for the detection

An indirect enzyme-linked immunosorbent assay (ELISA) was evaluated for the detection of Brucella antibodies in milk from water buffalo (Bubalus bubalis Linnaeus, 1758). The test accuracy was evaluated on milk samples from the Campania Region in Italy. A total of 100 negative samples were collected from 10 officially brucellosis-free herds in Salerno Province, while 30 positive samples were collected from 3 herds in Caserta Province, where animals gave positive results to the official tests and it was here that Brucella abortus biovar 1 had been isolated. Test sensitivity was 100%, with a confidence interval (CI) of 90.8%-100%, while specificity was 98% (CI 93%-99.4%) on individual milk samples. To simulate bulk milk samples from herds infected at various levels of infection, dilutions from 1:10 to 1:100 of positive milk samples in negative milk were also used. The probability of detecting antibodies in positive milk samples was higher than 50% up to a dilution of 1:50 in negative milk. Considering the average national water buffalo herd size, the probability of identifying infection in a water buffalo herd by bulk milk testing is 50% (CI 33.1%-66.9%) in the worst case scenario of a single infected animal contributing to the bulk milk

Uso della chemiluminescenza per la diagnosi della brucellosi bovina e ovina mediante ELISA indiretta e competitiva

I metodi ufficiali previsti dal Piano nazionale di eradicazione della brucellosi bovina ed ovi-caprina sono la sieroagglutinazione rapida (SAR) con l’antigene acidificato al Rosa Bengala e la fissazione del complemento (FDC). Nella attuale fase del piano di eradicazione non è infrequente imbattersi in risultati di difficile interpretazione ottenuti con i test ufficiali, pertanto è necessario poter disporre di test aggiuntivi che presentino livelli di specificità e di sensibilità più elevati. A questo scopo sono stati validati due metodi ELISA, indiretto (i-ELISA CL) e competitivo (c-ELISA CL), mediante l’utilizzo di un substrato chemiluminescente per la determinazione di anticorpi anti-Brucella in siero bovino ed ovino. I metodi si basano sulla rivelazione degli anticorpi anti-Brucella, contenuti nel siero, mediante la catalisi di un substrato enzimatico chemiluminescente (sistema luminolo/H2O2/ enhancer), da parte della perossidasi coniugata ad anticorpi secondari anti-IgG, nella i-ELISA CL, o al monoclonale anti-LPS, nella c-ELISA CL. Sulla base dei risultati ottenuti, per l'i-ELISA CL è stato stabilito un valore di cut-off, espresso come percentuale di positività (PP), del 60% per i sieri bovini e del 37,5% per quelli ovini; con questo valore di cut-off si ottiene una sensibilità ed una specificità del test del 100% per i sieri bovini, e una sensibilità del 100% e una specificità del 99,8% per quelli ovini. Per la c-ELISA CL è stato scelto un cut-off, espresso come percentuale di inibizione (PI), del 30% per i sieri bovini e del 40% per quelli ovini, che assicura valori di sensibilità e specificità del 100% in entrambi i casi

An ELISA for the evaluation of gamma interferon production in cattle vaccinated with Brucella abortus strain RB51

The results of an enzyme-linked immunosorbent assay (ELISA) implemented for the detection of gamma interferon (g-interferon) production in cattle vaccinated with Brucella abortus strain RB51 are presented. A purified protein fraction derived from RB51 (RB51 brucellin) has been used as antigenic stimulus for whole blood. The test was evaluated for 300 days in ten heifers vaccinated at calfhood with 10 × 109 colony-forming units of RB51 and in five control heifers. All animals came from officially brucellosis-free herds. Vaccinated animals started to give positive results from day 17 post vaccination (pv) until day 239 pv. All vaccinated animals gave a positive reaction at least once (with a stimulation index exceeding 2.5). Nevertheless, if sampling on day 20 pv is excluded (90% of vaccinated animals gave positive results), the sensitivity of the test varies from 20% to 70%, with a 40% average. A stimulation index over 2.5 was also recorded in three control animals. The results suggest that the g-interferon test is not suitable for the detection of cattle vaccinated with RB51, either at the individual or at the herd level

Valutazione della produzione di gamma interferone in bovini vaccinati con Brucella abortus ceppo RB51 mediante un test ELISA

In questo lavoro sono presentati i risultati di un test ELISA messo a punto per rilevare la produzione di gamma interferone (g-interferone) in bovini vaccinati con Brucella abortus ceppo RB51 (RB51). Come stimolo antigenico per il sangue intero è stata utilizzata una frazione proteica purificata derivante da RB51 (brucellina RB51). La prova è stata valutata nell’arco di 300 giorni in 10 manze vaccinate in età prepubere con 10×109 Unità Formanti Colonia di RB51 e in cinque manze di controllo, provenienti da allevamenti ufficialmente indenni da brucellosi bovina. I capi vaccinati hanno cominciato a fornire risultati positivi a partire dal 17° giorno post vaccinazione (p.v.) fino al giorno 239 p.v. Tutti i capi vaccinati hanno fornito almeno una volta un risultato positivo (indice di stimolazione, IS, superiore a 2,5). Tuttavia, se si esclude il prelievo al giorno 20 p.v. (90% di animali vaccinati risultati positivi), la sensibilità del test oscilla tra il 20% e il 70%, con una media del 40%. IS superiore a 2,5 è stato rilevato anche in tre animali di controllo. Sulla scorta dei risultati ottenuti, si ritiene che il test del g-interferone non fornisce garanzie sufficienti per consigliarne l’impiego ai fini di riconoscere i bovini vaccinati con RB51, sia come prova individuale, sia come prova d’allevamento

Serological screening of Coxiella burnetii (Q fever) and Brucella spp. in sheep flocks in the northern prefectures of Japan in 2007

Ovine sera collected from the northern Prefectures of Hokkaido, Iwate and Aomori in Japan, were examined for the presence of antibodies against Coxiella burnetii (Q fever) using the complement fixation test and, against Brucella spp., using both the rapid serum agglutination test and the complement fixation test. None of the sera tested were serologically positive to Brucella spp. A total of 21 animals (8.64%) out of 243 samples tested were seropositive to the C. burnetii antigen. Levels of infection were observed in all of the three Prefectures and in ten flocks of the fourteen sampled. Although no diagnostic measures were in place, the infection could not be linked to losses in sheep production or to the decreased fertility in ewes, a lower lambing rate and mortality in lambs. These data confirmed that Q fever is widespread in the sheep population in the area studied. Considering the zoonotic potential of the disease, further studies to investigate the epidemiology of Q fever in this region are required

Development and evaluation of diagnostic tests for the serological diagnosis of brucellosis in swine

A competitive enzyme-linked immunosorbent assay (c-ELISA), an indirect ELISA (i-ELISA) and a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) were developed to test for antibodies to Brucella suis in pig and wild boar sera. An anti-Brucella-LPS monoclonal antibody (MAb 4B5A) (c-ELISA and DELFIA) and an anti-swine IgG monoclonal antibody (MAb 10C2G5) (i-ELISA) were used for the three assays. The specificity (Sp) and sensitivity (Se) of the assays gave the following results: Se and Sp = 100% at a cut-off value of 61.0% (B/B0%) for c-ELISA; Sp = 99.1% and Se = 100% at a cut-off value of 21.7% (percentage positivity: PP%) for i-ELISA; Sp = 91.0% and Se = 75% at a cut-off value of 37.0% (B/B0%) for DELFIA. In addition, the performance of a commercial fluorescence polarisation assay (FPA), standardised for bovine sera, was evaluated in swine sera. The specificity and sensitivity obtained were both 100% at a cut-off value of 99.5 (millipolarisation unit values). These results suggest that the combination of c-ELISA, i-ELISA and FPA can be used to improve the serological diagnosis of swine brucellosis

Sviluppo e valutazione di test diagnostici per la sierodiagnosi di brucellosi suina

Sono stati sviluppati una ELISA competitiva (c-ELISA), una ELISA indiretta (i-ELISA) e un test immunologico DELFIA (Dissociation-Enhanced Lanthanide Fluorescence Immunoassay) per la ricerca di anticorpi verso Brucella suis in sieri di maiale e cinghiale. I tre test prevedono l’utilizzo di un anticorpo monoclonale (MAb 4B5A) verso l’LPS di Brucella (c-ELISA e DELFIA) e di un anticorpo monoclonale (MAb 10C2G5) verso le IgG suine (i-ELISA). La specificità (Sp) e la sensibilità (Se) dei tre test sono le seguenti: per la c-ELISA Se e Sp = 100% con un valore di cut-off pari al 61.0% (B/B0%); per la i-ELISA Sp = 99.1% e Se = 100% con un valore di cut-off di 21.7% (PP%); per il DELFIA Sp = 91.0% e Se = 75% ponendo il valore di cut-off al 37.0% (B/B0%). Inoltre sono state valutate le performance, nei confronti di sieri suini, di un test FPA (Fluorescence Polarization Assay) commerciale sviluppato per la ricerca di anticorpi anti-Brucella in sieri bovini; la specificità e la sensibilità ottenute sono entrambe del 100% al valore di cut-off di 99.5 (mP). Questi risultati suggeriscono che la combinazione di c-ELISA, i-ELISA e FPA può essere utilizzata per migliorare la diagnosi di brucellosi suina