Immunohistochemical investigations on Brucella ceti-infected, neurobrucellosis-affected striped dolphins (Stenella coeruleoalba)

Bacteria of the genus Brucella cause brucellosis, an infectious disease common to humans as well as to terrestrial and aquatic mammals. Since 1994 several cases of Brucella spp. infection have been reported in marine mammals worldwide. Indeed, since human brucellosis ranks as one of the most common bacterial zoonotic infections on a global scale, it is necessary to increase our knowledge about it also in the marine environment. Brucella ceti, which is phenotypically similar to other smooth brucellae as B. abortus and B. melitensis, shares with the latter two the same surface antigens that are routinely used for the serological diagnosis of Brucella spp. infection. Marine mammal Brucella spp. infections are characterized by a pathogenicity similar to their terrestrial counterparts, with the occurrence of abortion, stillbirth and orchitis and an involvement of the host’s central nervous system (CNS), similarly to what happens in mankind. While sero-epidemiological data suggest that Brucella spp. infection is widespread globally, detecting Brucella spp.-associated antigens by immunohistochemistry (IHC) in tissues from infected animals is often troublesome. The present study was aimed at investigating, by means of IHC based upon the utilization of an anti-Brucella LPS monoclonal antibody (MAb), the CNS immunoreactivity (IR) shown by B. ceti-infected, neurobrucellosis-affected striped dolphins

Development of a Capture ELISA for Rapid Detection of Salmonella enterica in Food Samples

In this study, an immunology-based assay that employed specificmonoclonal antibodies binding with somatic or flagella antigens of Salmonella enterica subsp. enterica was performed. As this pathogen is one of themost important bacterial species responsible for foodborne outbreaks, its detection in food by rapid and easy methods is properly suitable. After a first screening by indirect ELISA, three monoclonal antibodies (1B6D9, 1B6C11, 1D12F11) versus S. enterica subsp. enterica serovar TyphimuriumATCC 14028 (whole antigen) and another one (4E6F11) versus S. enterica flagellin were further characterized by immunoblotting and mass spectrometry analysis. Then, a total of 84 food samples (dairy products, meat, pasta and flour, eggs, and animal feed) were analyzed by both the officialmethod ISO6579:2002 and S. enterica capture ELISA. For the standardization of the lastmethod, the specific monoclonal antibody 4E6F11 was selected. The developed Salmonella capture ELISA showed a significant agreement with the official method (ISO 6579:2002). Relative sensitivity, specificity, and accuracy were 100%, 81.0%, and 90.5%, respectively. Therefore, this assay could represent a valid alternative to conventional methods able to detect this pathogen in foo

Development and validation of an antigen capture ELISA based on monoclonal antibodies specific for Listeria monocytogenes in food

A capture enzyme-linked immunosorbent assay (ELISA) for the identification of Listeria monocytogenes in food was standardised and validated. The assay was refined by analysing samples of meat, seafood, dairy products, pasta and flour. The method was found to be 100% specific for Listeria spp. tested, with a limit of sensitivity of 6.6 × 10(3) colony-forming units (cfu)/ml. Comparison of L. monocytogenes capture ELISA against the official International Organization for Standardization (ISO) method 11290-1:1996 for the isolation and identification of L. monocytogenes in food matrices produced a significant concordance index. The assay was validated on food matrices including meat, seafood and dairy products in line with ISO 16140:2003 concerning qualitative analytical methods. The assay was found to be accurate, specific, sensitive, selective, reproducible and fast, resulting in lower costs and faster turnaround in microbiological screening of foods

Sviluppo e validazione di un antigene-capture ELISA basato su anticorpi monoclonali specifici per Listeria monocytogenes negli alimenti

È stato standardizzato e validato un dosaggio immunoenzimatico capture ELISA per l’identificazione di Listeria monocytogenes negli alimenti. Il dosaggio è stato messo a punto analizzando campioni di prodotti carnei, ittici e lattiero-caseari, pasta di semola e di farina di grano. Il metodo è risultato specifico al 100% per Listeria spp., con limite di rivelazione di 6,6 × 10(3) cfu/ml. Il metodo L. monocytogenes capture ELISA è stato confrontato con il metodo ufficiale ISO 11290-1:1996 per l’isolamento e l’identificazione di L. monocytogenes in matrici alimentari ottenendo un indice di concordanza significativo. Il dosaggio è stato validato in base alle indicazioni della norma ISO 16140:2003 relativamente ai metodi di analisi qualitativi. Il dosaggio è risultato accurato, specifico, sensibile, selettivo, riproducibile e rapido da eseguire, consentendo nello screening degli alimenti la riduzione di tempi e costi dell’indagine microbiologica

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

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

Effetti del siero immune su colture cellulari di macrofagi infettati con Mycoplasma mycoides subsp. mycoides small colony: analisi morfologica mediante microscopia elettronica a scansione

I macrofagi sono cellule del sistema immunitario che svolgono un ruolo fondamentale nel meccanismo di difesa dell’ospite contro gli agenti patogeni. Attualmente è ancora poco chiara la dinamica del loro intervento e della risposta umorale nel favorire l’attività fagocitaria nei confronti di Mycoplasma mycoides subsp. mycoides small colony (Mmm-SC), agente eziologico della pleuropolmonite contagiosa bovina (PPCB). Questo studio è stato condotto al fine di valutare le modificazioni morfologiche della superficie macrofagica in seguito all’infezione in vitro con Mmm-SC in presenza di siero immune bovino. L’analisi morfologica dei macrofagi è stata condotta su colture, 6 ore dopo l’infezione, utilizzando l’analisi tridimensionale della microscopia elettronica a scansione. I macrofagi non infettati, in presenza di siero negativo o immune, e i macrofagi infettati con Mmm-SC, in assenza di siero, hanno mostrato solo lievi modificazioni della loro superficie. Al contrario, in colture di macrofagi infettati e in presenza di siero immune sono state osservate marcate modificazioni della superficie cellulare, come ampi veli e filopodi (indicatori di attivazione cellulare), e piccoli aggregati di micoplasmi in stretto contatto con le membrane macrofagiche. I risultati suggeriscono che la risposta umorale specifica per Mmm-SC può contribuire a innescare l’attività fagocitaria dei macrofagi

Effects of immune serum on macrophage cell cultures infected with Mycoplasma mycoides subsp. mycoides small colony: morphological analysis by scanning electron microscopy

Macrophages are pivotal cells of the immune system and play a key role in the host defence mechanism against pathogens. To date, the importance of macrophages and the role of humoral response in eliciting macrophage activity against Mycoplasma mycoides subsp. mycoides small colony (Mmm‑SC), the causative agent of contagious bovine pleuropneumonia (CBPP), have only been marginally elucidated or are almost unknown. The present study was undertaken to investigate the changes in surface morphology of macrophages after in vitro infection with Mmm‑SC in the presence of bovine immune serum. Morphological analysis was performed on macrophage cultures at 6 h post infection using the three-dimensional vision of scanning electron microscopy. Non-infected macrophages in the presence of negative or immune serum and macro phages infected with Mmm‑SC in the absence of serum showed only minor cell surface changes. In contrast, clear surface modifications, broad veils, fine philopodia highlighting cell activation and small aggregates of mycoplasma closely attached to the macrophage membrane, were observed in infected macrophage cultures in the presence of immune serum. Our results suggest that specific humoral response to Mmm‑SC may contribute and support phagocytic activity of macrophages

Proteomic analysis of Brucella melitensis and Brucella ovis for identification of virulence factor using bioinformatics approachs

The genus Brucella includes several genetically monomorphic species but with different phenotypic and virulence characteristics. In this study, proteins of two Brucella species, B. melitensis type strain 16M and B. ovis REO198 were compared by proteomics approach, in order to explain the phenotypic and pathophysiological differences among Brucella species and correlate them with virulence factors. Protein extracts from the two Brucella species were separated by SDS-PAGE and 5 areas, which resulted qualitatively and quantitatively different, were analyzed by nLC-MS / MS. A total of 880 proteins (274 proteins of B. melitensis and 606 proteins of B. ovis) were identified; their functional and structural features were analyzed by bioinformatics tools. Four unique peptides belonging to 3 proteins for B. ovis and 10 peptides derived from 7 proteins for B. melitensis were chosen for the high amount of predicted B-cell epitopes exposed to the solvent. Among these proteins, outer-membrane immunogenic protein (N8LTS7) and 25 kDa outer-membrane immunogenic protein (Q45321), respectively of B. ovis and B. melitensis, could be interesting candidates for improving diagnostics tests and vaccines. Moreover, 8 and 13 outer and periplasmic non homologue proteins of B. ovis and B. melitensis were identified to screen the phenotypic differences between the two Brucella strains. These proteins will be used to unravel pathogenesis and ameliorate current diagnostic assays