Development of a rapid method for the detection of Yersinia enterocolitica serotype O:8 from food

In this study, a new and alternative method based on monoclonal antibodies (MAbs) for the rapid detection of Yersinia enterocolitica O:8 was developed. This microorganism is an emerging foodborne pathogen causing gastrointestinal disease in humans. The transmission can occur through contaminated food such as raw or undercooked meat, milk and dairy products, water and fresh vegetables. Nine MAbs (46F7, 54B11, 54C11, 62D10, 64C7, 64C10, 72E8, 72E10, 72G6) were characterized and selected versus Y. enterocolitica O:8, and only 2 of them showed also a weak cross-reaction with Campylobacter jejuni. The MAb 54B11 was used for the development of Y. enterocolitica capture-ELISA in food matrices, i.e. meat and dairy products (n ¼ 132). The method was validated by ISO 16140:2003 and compared with the official method for the detection of presumptive pathogenic Y. enterocolitica (ISO 10273:2003). Relative accuracy, sensitivity and specificity corresponded to 100%. The selectivity was evaluated on other food samples (n ¼ 126) showing a lower confidence limit of 90.3% and an upper confidence limit of 100%. The results from this study demonstrated that the developed method was rapid and cheap, specific and sensitive for the screening of the pathogen in food

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

L’immunogenicità nella cavia e nel cavallo di due formulazioni di un vaccino inattivato e adiuvato per la peste equina

L’efficacia di due vaccini monovalenti, inattivati e adiuvati per il controllo della Peste Equina, allestiti con i sierotipi 5 e 9, è stata saggiata su cavia per selezionare la formulazione con le migliori capacità immunogene. Nella formulazione dei vaccini sono state prese in considerazione: la risposta immunitaria evocata nella cavia e le proprietà infiammatorie di due diversi tipi di adiuvanti precedentemente saggiati nella specie di destino del vaccino.Il vaccino allestito con il sierotipo 9, saggiato in uno studio pilota su cavallo, si è dimostrato capace fin dalla prima somministrazione di stimolare la produzione di anticorpi neutralizzanti. La risposta anticorpale evocata ha subito un marcato rialzo dopo la somministrazione della dose di richiamo, effettuata dopo 28 giorni, perdurando per almeno 10 mesi. La cavia sembra essere un utile modello di laboratorio per la valutazione delle proprietà antigeniche dei vaccini contro la peste equina

Immunogenicity of two adjuvant formulations of an inactivated African horse sickness vaccine in guinea-pigs and target animals

Monovalent, inactivated and adjuvanted vaccines against African horse sickness, prepared with serotypes 5 and 9, were tested on guinea-pigs to select the formulation that offered the greatest immunity. The final formulation of the vaccines took into account the immune response in the guinea-pig and the inflammatory properties of two types of adjuvant previously tested on target animals. A pilot study was subsequently conducted on horses using a vaccine prepared with serotype 9. The vaccine stimulated neutralising antibodies from the first administration and, after the booster dose, 28 days later; high antibody levels were recorded for at least 10 months. The guinea-pig appears to be a useful laboratory model for the evaluation of the antigenic properties of African horse sickness vaccines

Vaccino inattivato e adiuvato per il controllo delle infezioni da sierotipo 9 del virus della peste equina: valutazione dell'efficacia in cavallo e cavia

La peste equina (PE) è una malattia virale non contagiosa dei solipedi trasmessa da insetti vettori appartenenti al genere Culicoides. La malattia è endemica in numerose regioni dell'Africa e passate esperienze hanno evidenziato come l'Italia sia un paese esposto alle malattie infettive emergenti, endemiche in Africa. Un'incursione del virus della PE unitamente alla presenza del vettore Culicoides potrebbero essere causa di una emergenza epidemica. Onderstepoort Biological Products (OBP) commercializza un vaccino vivo attenuato contenente 7 dei 9 sierotipi virali, i sierotipi 5 e 9 sono esclusi dal vaccino. L'uso di tali vaccini è controverso, pertanto, da diversi anni, vengono condotti studi su prodotti inattivati o ricombinanti. Poiché la ricerca sui vaccini PE è ostacolata dalla necessità di utilizzare il cavallo per la valutazione dell'immunogenicità, in un precedente esperimento è stata studiata la risposta sierologica ai sierotipi 5 e 9, in cavie e cavalli. Nelle due specie animali è stata osservata una risposta durevole e sovrapponibile. Nel presente studio sono state saggiate per un periodo di 12 mesi le risposte sierologiche ottenute in cavie e cavalli immunizzati con il vaccino inattivato formulato con il sierotipo 9. Le risposte sierologiche nelle due specie animali si sono confermate sovrapponibili. Al challenge dell'immunità i cavalli sono risultati protetti dall'infezione e dalla malattia

Inactivated and adjuvanted vaccine for the control of the African horse sickness virus serotype 9 infection: evaluation of efficacy in horses and guinea-pig model

African horse sickness (AHS) is a non-contagious viral disease of solipeds transmitted by Culicoides. The disease is endemic in most African countries. Past experience has shown that Italy is a country exposed to emerging infectious diseases endemic to Africa; an incursion of AHS virus together with the widespread presence of Culicoides vectors could be the cause of a serious epidemic emergency. A live attenuated vaccine containing seven of the nine viral serotypes, serotype 5 and 9 are excluded, is commercially available from Onderstepoort Biological Products. However, the use of live vaccines is a matter of endless disputes, and therefore inactivated or recombinant alternative products have been investigated over the years. Since research on AHS is hampered by the use of horses to evaluate vaccine potency, in a previous experiment serological response to serotypes 5 and 9 was assayed in guinea-pigs and horses. A durable and comparable serological response was observed in the two animal species. In the present study antibody response in horses and guinea-pigs, immunised with the inactivated-adjuvanted vaccine formulated with serotype 9, was tested over a period of 12 months. When immunity was challenged, horses were protected from infection and disease. Antibody response in horses and guinea-pigs compared favourably

Determinants of Bluetongue Virus Virulence in Murine Models of Disease ▿

Bluetongue is a major infectious disease of ruminants that is caused by bluetongue virus (BTV). In this study, we analyzed virulence and genetic differences of (i) three BTV field strains from Italy maintained at either a low (L strains) or high (H strains) passage number in cell culture and (ii) three South African “reference” wild-type strains and their corresponding live attenuated vaccine strains. The Italian BTV L strains, in general, were lethal for both newborn NIH-Swiss mice inoculated intracerebrally and adult type I interferon receptor-deficient (IFNAR−/−) mice, while the virulence of the H strains was attenuated significantly in both experimental models. Similarly, the South African vaccine strains were not pathogenic for IFNAR−/− mice, while the corresponding wild-type strains were virulent. Thus, attenuation of the virulence of the BTV strains used in this study is not mediated by the presence of an intact interferon system. No clear distinction in virulence was observed for the South African BTV strains in newborn NIH-Swiss mice. Full genomic sequencing revealed relatively few amino acid substitutions, scattered in several different viral proteins, for the strains found to be attenuated in mice compared to the pathogenic related strains. However, only the genome segments encoding VP1, VP2, and NS2 consistently showed nonsynonymous changes between all virulent and attenuated strain pairs. This study established an experimental platform for investigating the determinants of BTV virulence. Future studies using reverse genetics will allow researchers to precisely map and “weight” the relative influences of the various genome segments and viral proteins on BTV virulence

Analysis of <i>Trypanosoma equiperdum</i> Recombinant Proteins for the Serological Diagnosis of Dourine

The significance of Trypanosoma equiperdum as the causative agent of dourine cannot be understated, especially given its high mortality rate among equids. International movement of equids should be subject to thorough health checks and screenings to ensure that animals are not infected with Trypanosoma equiperdum. This involves the implementation of quarantine protocols, testing procedures, and the issuance of health certificates to certify the health status of the animals. Three proteins, the peptidyl-prolyl cis-trans isomerase (A0A1G4I8N3), the GrpE protein homolog (A0A1G4I464) and the transport protein particle (TRAPP) component, putative (A0A1G4I740) (UniProt accession numbers SCU68469.1, SCU66661.1 and SCU67727.1), were identified as unique to T. equiperdum by bioinformatics analysis. The proteins were expressed as recombinant proteins and tested using an indirect ELISA and immunoblotting test with a panel of horse positive and negative sera for dourine. The diagnostic sensitivity, specificity and accuracy of the i-ELISAs were 86.7%, 53.8% and 59.0% for A0A1G4I8N3; 53.3%, 58.7% and 57.9% for A0A1G4I464; and 73.3%, 65.0% and 66.3% for A0A1G4I740, respectively, while the diagnostic sensitivity, specificity and accuracy of immunoblotting were 86.7%, 92.5% and 91.6% for A0A1G4I8N3; 46.7%, 81.3% and 75.8% for A0A1G4I464; and 80.0%, 63.8% and 66.3% for A0A1G4I740. Among the three proteins evaluated in the present work, A0A1G4I8N3 provided the best results when tested by immunoblotting; diagnostic application of this protein should be further investigated using a greater number of positive and negative sera

Efficacy of an inactivated EHDV-8 vaccine in preventing viraemia and clinical signs in experimentally infected cattle

Epizootic haemorrhagic disease (EHD), caused by the EHD virus (EHDV), is a vector-borne viral disease transmitted through Culicoides biting midges. EHDV comprises seven serotypes (1, 2, and 4–8), with EHDV-8 having recently emerged and spread in Europe over the last two years. Such event has raised concerns about the significant threat posed by EHDV-8 to livestock industry. In this study, an inactivated vaccine against EHDV-8 (vEHDV8-IZSAM) was developed. Safety and efficacy of the vaccine were evaluated in calves through clinical, serological, and virological monitoring following experimental challenge.The vaccine was proven safe, with only transient fever and localized reactions observed in a few animals, consistent with adjuvanted vaccine side effects. vEHDV8-IZSAM elicited a robust humoral response, as evidenced by the presence of neutralizing antibodies. After challenge with a virulent isolate, viraemia and clinical signs were evidenced in control animals but in none of the vaccinated animals.This study highlights the potential of vEHDV8-IZSAM as a safe and highly effective vaccine against EHDV-8 in cattle. It offers protection from clinical disease and effectively prevents viraemia. With the recent spread of EHDV-8 in European livestock, the use of an inactivated vaccine could be key in protecting animals from clinical disease and thus to mitigate the economic impact of the disease. Further investigations are warranted to assess the duration of the induced immunity and the applicability of this vaccine in real-world settings. Accordingly, joint efforts between public veterinary institutions and pharmaceutical companies are recommended to scale up vaccine production