Western Bluetongue virus serotype 3 in Sardinia, diagnosis and characterization.

Over the last 20 years, Italy has experienced multiple incursions of different serotypes of Bluetongue virus (BTV), a Culicoides-borne arbovirus, the causative agent of bluetongue (BT), a major disease of ruminants. The majority of these incursions originated from Northern Africa, likely because of wind-blown dissemination of infected midges. Here, we report the first identification of BTV-3 in Sardinia, Italy. BTV-3 circulation was evidenced in sentinel animals located in the province of Sud Sardegna on September 19, 2018. Prototype strain BTV-3 SAR2018 was isolated on cell culture. BTV-3 SAR2018 sequence and partial sequences obtained by next-generation sequencing from nucleic acids purified from the isolate and blood samples, respectively, were demonstrated to be almost identical (99-100% of nucleotide identity) to BTV-3 TUN2016 identified in Tunisia in 2016 and 2017, a scenario already observed in past incursions of other BTV serotypes originating from Northern Africa.

A novel Bluetongue virus serotype 3 strain in Tunisia, November 2016.

Since 1998, southern Europe has experienced multiple incursions of different serotypes and topotypes of Bluetongue virus, a vector-borne transmitted virus, the causative agent of Bluetongue (BT), a major disease of ruminants. Some of these incursions originated from northern Africa, likely because of wind-blown dissemination of infected midges. In this report, we describe the detection and whole genome characterization of a novel BTV-3 strain identified in a symptomatic sheep in Tunisia. Sequences were immediately deposited with the GenBank Database under Accession Nos KY432369-KY432378. Alert and preparedness are requested to face the next vector seasons in northern Africa and the potential incursion of this novel strain in southern Europe.

Bluetongue virus in Lebanon.

Since 2000, several incursions of bluetongue virus (BTV) occurred in the Mediterranean Basin involving European and surrounding Countries. The Middle East represents one of the most important gateways for the access of BTV in Europe. Limited data on the BTV situation in this area are available. In this perspective, an epidemiological survey on the presence of BTV in Lebanon was conducted. Of the 181 serum samples tested, 97 (mean = 53.6%; 95% CI: 46.3-60.7) resulted positive when tested for the presence of BTV antibodies by c-ELISA, of these 42 (mean = 42%; 95% CI: 32.8-51.8) serum samples were from sheep and 55 (mean = 67.9%; 95% CI: 57.1-77.1) serum samples were from goats. Fourteen blood samples (14/110; mean = 12.7%; 95% CI: 7.8-20.3), 6 (6/66; mean = 9.1%; 95% CI: 4.4-18.5) from sheep and 8 (8/44; mean = 18.2%; 95% CI: 9.6-32.0) from goats, were positive by qRT-PCR. The results with serum-neutralization assay and typing performed by RT-PCR confirmed that six BTV serotypes are currently circulating in Lebanon, and these serotypes are as follows: 1, 4, 6, 8, 16 and 24. This study is the first report that confirms the presence and circulation of BTV in Lebanon.

Development and validation of a competitive ELISA kit for the serological diagnosis of ovine, caprine and bovine brucellosis.

A competitive ELISA (Brucella-Ab c-ELISA) was standardized and validated for the detection of Brucella antibodies in cattle, sheep and goat sera using a monoclonal antibody (MAb 4B5A) produced against Brucella melitensis biotype 2. The specificity and sensitivity of the assay were 100% to a 67.5% cut-off point (B/Bo%). When compared with an indirect ELISA, the Brucella-Ab c-ELISA did not demonstrate cross-reactions when testing positive sera for antibodies to some Enterobacteriaceae. A comparison was made between the Brucella-Ab c-ELISA and the complement fixation and Rose Bengal tests. Results demonstrated that the Brucella-Ab c-ELISA is a valuable tool for the serological diagnosis of bovine and ovine/caprine brucellosis.

Production and characterisation of monoclonal antibodies specific for Escherichia coli O157:H7.

Seven monoclonal antibodies (MAbs) specific for Escherichia coli O157:H7, one of the major causes of haemorrhagic colitis in humans, were produced by immunising Balb/c mice with the strain E. coli O157:H7. These monoclonal antibodies do not cross-react with other bacteria such as Salmonella enterica serovar Typhimurium, E. coli O14, E. coli JM109, S. enterica serovar Enteritidis, S. panama, S. saintpaul, S. derby, S. muenchen, S. bredeney, S. hadar, Yersinia enterocolitica, Proteus vulgaris, Shigella flexneri, Listeria ivanovii, L. monocytogenes 13M, L. innocua, Enterobacter cloacae, E. agglomerans, E. amnigenus, Citrobacter freundii, Escherichia fergussoni or Klebsiella pneumoniae. Of the seven MAbs obtained, MAb 8B8C3 was selected to prepare a high-sensitivity sandwich ELISA method specific for O157:H7.

Rapid detection of bluetongue virus in blood and organ samples using a capture enzyme-linked immunosorbent assay.

An antigen capture ELISA for bluetongue (BT) virus was developed using tissue culture supernatant to identify different BT virus (BTV) serotypes 1, 2, 4, 9 and 16, which have been incriminated in the current epidemic in the Mediterranean Basin. To obtain a positive result and after amplification in tissue culture, the minimum amount of infecting virus required was 100 TCID(50). Results from the antigen capture ELISA were compared with conventional methods for virus isolation and identification, such as virus amplification on embryonated chicken eggs (ECEs), followed by tissue culture and the direct immunofluorescence test. The sensitivity and specificity of the ELISA in infected tissue culture supernatant using homogenates of BTV-positive ovine and bovine organs and blood, without a previous step in ECEs, were 100%. The assay was also applied to homogenates of chicken embryo tissues, which had been infected with different BTV serotypes. This method enabled detection of the virus with 100% sensitivity and specificity rates, also using amplification in ECEs. Furthermore, among the various embryo tissues tested, liver was found to be the most suitable for use with ELISA. In experimentally infected ovine blood samples, the ELISA revealed the presence of the virus. Given the high sensitivity and specificity obtained with the BTV serotypes in this trial, the method should greatly facilitate BT diagnosis.

Bluetongue laboratory diagnosis: a ring test to evaluate serological results using a competitive ELISA kit.

The occurrence of bluetongue (BT) in Italy prompted an increase in disease surveillance. Thus a competitive enzyme-linked immunosorbent assay (c-ELISA) to detect immunoglobulins to BT virus (BTV) was developed and distributed amongst 27 laboratories comprising the Italian veterinary diagnostic laboratories network to screen field sera. This ring test enabled comparison of the results and the evaluation of the reproducibility of the method. The c-ELISA developed by the National Reference Centre for Exotic Diseases (c-ELISA-IZSA&M) was compared also against a commercially available c-ELISA. In addition, results obtained by the Centre of Athens Veterinary Institutions are presented.

A reporter protein for meat integrity.

A method for distinguishing fresh from thawed frozen samples of porcine and bovine meat is proposed.Crude soluble extracts from fresh (stored at +4 °C for 0-3 days) or deep frozen (at -80 °C) meat samples from pork and beef, were assayed by blocking ELISA to estimate m-MDH (mitochondrial malate dehydrogenase) titres. As a rule, a separate aliquot of each test sample was placed at -20 °C until frozen and subsequently thawed for the preparation of the extract whose analysis gave the 'self reference' m-MDH titre. For a given meat sample calculation of the tau index, 'τ', was proposed as the ratio between the self reference value and the original m-MDH titre. The 95% tolerance for τ for fresh and thawed meat, suggests that the method is suitable for this kind of analysis.