Massimo Spedicato

Novel putative Bluetongue virus in healthy goats from Sardinia, Italy

In recent years, novel Bluetongue virus (BTV) serotypes have been isolated and/or sequenced by researchers within the field. During Bluetongue surveillance activities, we identified a putative novel BTV serotype in healthy goats from Sardinia, Italy. RNAs purified from blood and serum samples were positive for BTV by a generic real time RT-PCR and c-ELISA, respectively, whereas genotyping and serotyping were unsuccessful. By NGS, the whole genome sequence was obtained from two blood samples (BTV-X ITL2015 strains 34200 and 33531). Overall, Seg 2 of BTV-X ITL2015 shows the highest identity (75.3-75.5% nt/77.4-78.1% aa) with recently isolated BTV-27s from Corsica and with the last discovered BTV XJ1407 from China (75.9% nt /78.2% aa), whereas it is less related with BTV-25 from Switzerland (73.0% nt/75.0% aa) and BTV-26 from Kuwait (62.0% nt/60.5% aa). A specific RT-qPCR targeting Seg 2 of BTV-X ITL2015 was assessed in this study. Considering the Seg 2/VP2 identity of BTV-X ITL2015 with BTV-25, 26, 27s and BTV XJ1407 and that serum of BTV-X ITL2015 infected goats failed to neutralize all tested extant serotypes, we propose the existence of a novel BTV serotype circulating in goats in Sardinia. Isolation was so far unsuccessful thus hampering proper antigenic characterization.

Bluetongue virus surveillance in the Islamic Republic of Mauritania: Is serotype 26 circulating among cattle and dromedaries?

In March 2013, EDTA-blood and serum samples were collected from 119 cattle and 159 dromedaries at the slaughterhouse of Nouakchott, the capital city of the Islamic Republic of Mauritania. Serum samples were screened for the presence of Bluetongue (BT) antibodies by competitive ELISA (cELISA). Positive samples were then tested by serum-neutralization (SN) to determine BTV serotype. RNA from blood samples was first tested by a genus-specific quantitative RT-PCR assay which is able to detect all 27 existing BTV serotypes (RT-qPCR1-27). Positive samples were further screened by a RT-qPCR assay which, instead, is able to detect the classical 24 BTV serotypes only (RT-qPCR1-24). Of the 278 serum samples tested, 177 (mean=63.7%; 95% CI: 57.9%-69.1%) resulted positive by cELISA. Of these, 69 were from cattle (mean=58.0%; 95% CI: 49.0%-66.5%) and 108 from dromedaries (mean=67.9%; 95% CI: 60.3%-74.7%). BTV-26 neutralizing antibodies were by far the most frequently found as they were detected in 146 animals with titres ranging from 1:10 to 1:80. Out of 278 blood samples, 25 (mean=9.0%; 95% CI: 6.2%-12.9%) were found positive for BTV by RT-qPCR1-27, 20 (mean=16.8%; 95% CI: 11.2%-24.6%) were from cattle and 5 (mean=3.1%; 95% CI: 1.4%-7.1%) from dromedaries. When tested by RT-qPCR1-24 the 25 BTV positive samples were negative. Unfortunately, no genetic information by molecular typing or by next generation sequencing has been obtained as for the very low levels of RNA in the blood samples.

Analysis of bluetongue serotype 3 spread in Tunisia and discovery of a novel strain related to the bluetongue virus isolated from a commercial sheep pox vaccine

Bluetongue (BT), is one of the OIE-listed major diseases of ruminants. Following the official report of BT virus serotype 3 (BTV-3) in a sheep in Cap Bon (Tunisia), blood and serum samples of ruminants were collected from some areas of Tunisia to further investigate the presence of this virus in the country. A quantitative real time RT-PCR has been first developed for the detection and quantitation of BTV-3 RNA from field specimens. Out of 62 collected blood samples, 23 were shown to be positive for BTV-3 RNA. Isolation on cell cultures was also possible from six samples. Genome sequencing revealed the circulation of two unrelated western strains of BTV-3, one circulating in Cap Bon and neighboring areas, and the other circulating nearby the border with Libya. The presence of a putative novel BTV serotype (BTV-Y TUN2017) in sheep introduced from Libya to Tunisia, genomically related to the BTV strain contaminating a commercially-available sheep pox vaccine and to BTV-26, has been also demonstrated. This finding highlights the pressing need for a prompt production and release of a novel inactivated BTV-3 vaccine to be used in case of emergence or proactively in the areas of Southern Europe at major risk of BTV introduction. The assessment of a novel vaccine will certainly exalt the role and importance of surveillance activities and collaboration with Northern African countries.

Experimental infection of rock pigeons ( Columba livia ) with three West Nile virus lineage 1 strains isolated in Italy between 2009 and 2012

West Nile virus (WNV) circulation dynamics in the context of the urban environment is not yet elucidated. In this perspective, three groups of eight rock pigeons (Columbia livia) were inoculated with three WNV lineage 1 strains isolated in Italy between 2009 and 2012. The pigeons did not develop any clinical signs consistent with WNV acute infection. All animals seroconverted and shed virus up to 15 days post-infection by the oral or cloacal routes. In all infected groups viraemia lasted for 4 days post-infection. No WNV-specific gross or histological lesions were found in infected birds compared to control birds and immunohistochemistry remained constantly negative from all tissues. The reservoir competence index was also assessed and it ranged between 0·11 and 0·14. This study demonstrates that pigeons are competent reservoir hosts for Italian WNV lineage 1 circulating strains thus potentially posing a risk to the public health system.

Efficacy of vaccination for bluetongue virus serotype 8 performed shortly before challenge and implications for animal trade

Vaccination is the most effective strategy for controlling Bluetongue virus (BTV) spread and economic consequences thereof. In this study we verified in sheep, using one commercially available inactivated vaccine for BTV-8 (BTVPUR AlSap 8), when, during the recommended vaccination schedule, animals start to be effectively protected against challenge with wild-type strain. To this aim, sheep were challenged at different time points shortly after the first vaccine injection. Twenty-four Sarda sheep were divided into four groups vaccinated two weeks before challenge (Group A), one week before challenge (Group B) and concurrently with challenge (Group C). A second vaccine was performed twenty-eight days later with respect the first vaccine administration in each experimental group. The last group consisted of six non vaccinated-infected animals (NVIA). Virological and serological examinations were performed before and after challenge up to 42 and 77days post challenge, respectively. The results of the study show that vaccination commenced as little as two weeks before challenge (Group A) prevented viremia and RNAemia in challenged sheep altogether. Conversely, Group B was partially protected from challenge and Group C showed viraemia and RNAemia similar to NVIA. This study indicates that the first administration of inactivated vaccine performed two weeks before challenge was able to prevent viraemia. Overall, our findings may have direct consequences for the management of an unexpected BTV-8 outbreak in sheep and for the legislation on sheep trade from BTV restriction areas.

Innocuity of a commercial live attenuated vaccine for epizootic hemorrhagic disease virus serotype 2 in late-term pregnant cows

Epizootic hemorrhagic disease (EHD) is an arthropod-borne infectious viral disease sustained by the epizootic hemorrhagic disease virus (EHDV). The only commercially available and currently used vaccines are manufactured for EHDV-2 in Japan, either live or inactivated vaccines. In this study we tested the innocuity for fetuses of the live attenuated EHDV-2 vaccine in five late-term pregnant cows. Whole blood and serum samples were collected from dams and screened for the presence of EHDV-2 RNA, infectious virus and antibodies. After calving, whole blood and serum samples collected from calves, before and after colostrum intake, were also tested for antibodies and for virus detection. In dams, neither fever nor clinical signs were observed. All of them seroconverted and a strong humoral response was detected throughout the sampling period. All blood samples tested negative for EHDV-2 except for one sample collected from a dam 11 days post-vaccination which tested positive at virus isolation at the third cell passage following two rounds of blind passages. Although they had free access to colostrum, calves tested serologically negative for EHDV-2 during the entire course of the experiment. Overall, the tested live attenuated vaccine can be safely administered to late-term pregnant cows as it was not demonstrated to cross the placental barrier. The safety of the live-attenuated vaccine is further confirmed by the emergence of Ibaraki virus in 2013 in Japan which is apparently not related to the spread of the vaccine strain currently used in Japan.

Outbreak of porcine epidemic diarrhoea virus (PEDV) in Abruzzi region, central-Italy

Abstract Here we report and characterize a porcine epidemic diarrhea (PED) outbreak which occurred in a swine fattening farm in the province of Teramo, Abruzzi region (central Italy), in January 2016. PED virus (PEDV) identification was determined by real‐time RT‐PCR performed on RNAs purified from fecal samples collected from two symptomatic pigs. Whole genome sequence (PEDV 1842/2016) was also obtained by next generation sequencing straight from RNA purified from one fecal sample. Genome comparison with extant global PEDV strains revealed a high nucleotide identity with recently reported European and American S‐INDEL PEDVs. Efficient sequencing, share of genomic data combined with the implementation of epidemiological tools would be the ideal approach for study and analysis of transboundary infectious diseases as PED.

Prevalence and risk factors for bluetongue in the State of São Paulo, Brazil

Abstract Bluetongue (BT), caused by Bluetongue virus (BTV), is a disease that affects ruminants such as cattle, sheep, goats and deer. BTV is transmitted by female midges of the genus Culicoides. In Brazil, information on the prevalence of BTV in cattle is limited, so the objective of this work was to identify BTV serotypes in cattle. The State of São Paulo was divided into seven cattle‐producing regions, and in each of them, 300 cattle farms were randomly selected. One animal from each farm (out of a total of 1,598 farms) was selected and its sera tested by virus neutralization technique against BTV serotypes (1–24 and 26) for determining antibody titre. Moreover, for each sampled farm, an epidemiological questionnaire was submitted to verify the type of cattle production and the zootechnical and sanitary practices carried out, which could be associated with a higher risk of BTV infection. In this study, antibodies (percentage, [95% confidence interval]) were identified against 11 serotypes: BTV‐1 (22.15%, [15.72–27.92]), BTV‐2 (31.03%, [26.65–37.98]), BTV‐3 (18.96%, [12.42–24.90]), BTV‐4 (24.90% [19.41–29.12]), BTV‐9 (6.82%, [1.45–11.72]), BTV‐12 (7.50%, [2.82–12.51]), BTV‐17 (23.90%, [17.35–29.35]), BTV‐19 (10.20%, [4.62–5.56]), BTV‐21 (30.66%, [25.00–36.00]), BTV‐22 (12.14%, [5.91–18.55]), BTV‐26 (57.00%, [51.41–63.59]). In this study, for the first time in Brazil serological evidence of the presence of serotypes BTV‐2, BTV‐9, BTV‐21 and BTV‐26 is reported. The variable ‘new cattle entering herd’ was considered a risk factor for the occurrence of infection (OR = 2.183, 95% CI = 1.6–2.9).